Molecular polymorphisms of house dust mite allergens

Previous studies from this laboratory have described the primary amino acid sequences of the group I and group II allergens fromDermatophagoides pteronyssinus andD. farinae. This report concentrates on polymorphisms of allergens within a species. Firstly, four cDNA clones ofDer fII produced by polymerase chain reaction have been sequenced and are compared to the sequences published previously by ourselves and others. Although the sequences come from different sources, Australia and Japan, the overriding conclusion is one of similarity, with only two possible non-conservative changes in the six sequences. The nucleotides were also very conserved including the 3 untranslated regions, although some non-coding differences could be found which may provide a genetic marker. Experiments are reported to help define the group IIID. pteronyssinus allergens. Previous studies have characterised the group III ofD. farinae as a Mr 29-kDa molecule which can be defined by monoclonal antibodies. A Mr 17-kDa molecule ofD. pteronyssinus has been reported with an almost identical N-terminal sequence. Here it is described thatDer fIII isolated from different preparations of spent mite media by affinity chromatography have predominantly Mr 32-, 28- and 21-kDa forms which vary in degree from batch to batch. 83% of adults and 38% of children react with the preparation by radioimmune dot-blot. The difference between the children and adults is statistically significant and reactivity can be to at least the 32- and 28-kDa form. Antisera produced in mice against theDer fIII react toD. pteronyssinus mite extract by Western blotting primarily to a 32-kDa moiety, but also 28- and 21-kDa forms in some extracts.     

Real-time PCR detection of environmental mycobacteria in house dust

Analysing the number and species of microbes in indoor dust is needed for assessment of human exposure to microbes in dwellings. Environmental mycobacteria are common heterotrophic bacteria in both natural and man-made environments and potential inducers of human immune system. Culture of mycobacteria from samples rich with other microbes is difficult due to the slow growth rate of mycobacteria and this has hampered the studies on their role in indoor human exposure. A quantitative, real-time 5'-nuclease (TaqMan) PCR assay was developed to detect environmental mycobacteria in indoor dust samples. The specificity of the primers and the probe targeting the 16S rDNA of mycobacteria, tested with 26 mycobacterial and 10 non-mycobacterial but related species, proved to be high. When tested on 20 indoor dust samples collected from five homes, the assay gave counts varying between 4.8 × 10⁴ and 7.2 × 10⁶ cell/g, being on average 1.1 × 10³ times higher than culture. Seasonal variation in the dust counts of mycobacteria was observed by both culture and qPCR. Total of 140 isolates considered as mycobacteria by Ziehl-Neelsen staining and GLC-analyses were subjected to PCR analysis with the mycobacterial primers, and 39 isolates to partial 16S rDNA sequencing. All proved to be mycobacteria and revealed high diversity of mycobacterial species in the dust samples. Majority of the sequences were related to M. terrae and M. avium complexes.     

The castor-oil plant in the United States

About 125,000,000 pounds of castor oil are annually used in the United States in the manufacture of paints and varnishes, fatty acids, sulphonated oils, soap and other important products, 95% of which is imported either as seed or expressed oil. Attempts are now being made to revive a former American industry in the growing of domestic crops.     

Molecular identification of house dust mites and storage mites

Mites are known causes of allergic diseases. Currently, identification of mites based on morphology is difficult if only one mite is isolated from a (dust) sample, or when only one gender is found, or when the specimen is not intact especially with the loss of the legs. The purpose of this study was to use polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) of the ITS2 gene, to complement the morphological data for the identification of mites to the species level. For this, six species were cultured: Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis, Tyrophagus putrescentiae, Aleuroglyphus ovatus and Glycycometus malaysiensis. Genomic DNA of the mites was extracted, quantified, amplified and digested individually with restriction enzymes. Hinf I and Ple I differentiated the restriction patterns of D. pteronyssinus and D. farinae. Bfa I and Alu I enzymes differentiated B. tropicalis and G. malaysiensis. Ple I enzyme was useful for the differentiation between T. putrescentiae and A. ovatus. Bfa I was useful for the differentiation of G. malaysiensis from the rest of the species. In conclusion, different species of mites can be differentiated using PCR–RFLP of ITS2 region. With the established PCR–RFLP method in this study, identification of these mites to the species level is possible even if complete and intact adult specimens of both sexes are not available. As no study to date has reported PCR–RFLP method for the identification of domestic mites, the established method should be validated for the identification of other species of mites that were not included in this study.     

Mapping Atmospheric Moisture Climatologies across the Conterminous United States

Spatial climate datasets of 1981–2010 long-term mean monthly average dew point and minimum and maximum vapor pressure deficit were developed for the conterminous United States at 30-arcsec (~800m) resolution. Interpolation of long-term averages (twelve monthly values per variable) was performed using PRISM (Parameter-elevation Relationships on Independent Slopes Model). Surface stations available for analysis numbered only 4,000 for dew point and 3,500 for vapor pressure deficit, compared to 16,000 for previously-developed grids of 1981–2010 long-term mean monthly minimum and maximum temperature. Therefore, a form of Climatologically-Aided Interpolation (CAI) was used, in which the 1981–2010 temperature grids were used as predictor grids. For each grid cell, PRISM calculated a local regression function between the interpolated climate variable and the predictor grid. Nearby stations entering the regression were assigned weights based on the physiographic similarity of the station to the grid cell that included the effects of distance, elevation, coastal proximity, vertical atmospheric layer, and topographic position. Interpolation uncertainties were estimated using cross-validation exercises. Given that CAI interpolation was used, a new method was developed to allow uncertainties in predictor grids to be accounted for in estimating the total interpolation error. Local land use/land cover properties had noticeable effects on the spatial patterns of atmospheric moisture content and deficit. An example of this was relatively high dew points and low vapor pressure deficits at stations located in or near irrigated fields. The new grids, in combination with existing temperature grids, enable the user to derive a full suite of atmospheric moisture variables, such as minimum and maximum relative humidity, vapor pressure, and dew point depression, with accompanying assumptions. All of these grids are available online at http://prism.oregonstate.edu, and include 800-m and 4-km resolution data, images, metadata, pedigree information, and station inventory files.     

Spatial Scaling of Non-Native Fish Richness across the United States

A major goal and challenge of invasion ecology is to describe and interpret spatial and temporal patterns of species invasions. Here, we examined fish invasion patterns at four spatially structured and hierarchically nested scales across the contiguous United States (i.e., from large to small: region, basin, watershed, and sub-watershed). All spatial relationships in both richness and fraction between species groups (e.g., natives vs. exotics) were positive at large scales. However, contrary to predictions using null/neutral models, the patterns at small scales were hump-shaped (unimodal), not simply negative. The fractions of both domestic (introduced among watersheds within the USA) and foreign (introduced from abroad) exotics increased with area across scales but decreased within each scale. The foreign exotics exhibited the highest dominance (lowest evenness) and spatial variation in distribution, followed by domestic exotics and natives, although on average natives still occupy larger areas than domestic and foreign exotics. The results provide new insight into patterns and mechanisms of fish species invasions at multiple spatial scales in the United States.     

New reports of nuclear DNA content for 407 vascular plant taxa from the United States

Background and Aims

The amount of DNA in an unreplicated haploid nuclear genome (C-value) ranges over several orders of magnitude among plant species and represents a key metric for comparing plant genomes. To extend previously published datasets on plant nuclear content and to characterize the DNA content of many species present in one region of North America, flow cytometry was used to estimate C-values of woody and herbaceous species collected in Wisconsin and the Upper Peninsula of Michigan, USA.

Methods

A total of 674 samples and vouchers were collected from locations across Wisconsin and Michigan, USA. From these, C-value estimates were obtained for 514 species, subspecies and varieties of vascular plants. Nuclei were extracted from samples of these species in one of two buffers, stained with the fluorochrome propidium iodide, and an Accuri C-6 flow cytometer was used to measure fluorescence peaks relative to those of an internal standard. Replicate extractions, coefficients of variation and comparisons to published C-values in the same and related species were used to confirm the accuracy and reliability of our results.

Key Results and Conclusions

Prime C-values for 407 taxa are provided for which no published data exist, including 390 angiosperms, two gymnosperms, ten monilophytes and five lycophytes. Non-prime reports for 107 additional taxa are also provided. The prime values represent new reports for 129 genera and five families (of 303 genera and 97 families sampled). New family C-value maxima or minima are reported for Betulaceae, Ericaceae, Ranunculaceae and Sapindaceae. These data provide the basis for phylogenetic analyses of C-value variation and future analyses of how C-values covary with other functional traits.     

Molecular zoogeography of freshwater fishes in the southeastern United States

Restriction fragment length polymorphisms in mitochondrial DNA (mtDNA) were used to reconstruct evolutionary relationships of conspecific populations in four species of freshwater fish—Amia calva, Lepomis punctatus, L. gulosus, and L. microlophus. A suite of 14-17 endonucleases was employed to assay mtDNAs from 305 specimens collected from 14 river drainages extending from South Carolina to Louisiana. Extensive mtDNA polymorphism was observed within each assayed species. In both phenograms and Wagner parsimony networks, mtDNA clones that were closely related genetically were usually geographically contiguous. Within each species, major mtDNA phylogenetic breaks also distinguished populations from separate geographic regions, demonstrating that dispersal and gene flow have not been sufficient to override geographic influences on population subdivision.—Importantly, there were strong patterns of congruence across species in the geographic placements of the mtDNA phylogenetic breaks. Three major boundary regions were characterized by concentrations of phylogenetic discontinuities, and these zones agree well with previously described zoogeographic boundaries identified by a different kind of data base—distributional limits of species—suggesting that a common set of historical factors may account for both phenomena. Repeated episodes of eustatic sea level change along a relatively static continental morphology are the likely causes of several patterns of drainage isolation and coalescence, and these are discussed in relation to the genetic data.—Overall, results exemplify the positive role that intraspecific genetic analyses may play in historical zoogeographic reconstruction. They also point out the potential inadequacies of any interpretations of population genetic structure that fail to consider the influences of history in shaping that structure.     

Percutaneous Coronary Intervention Utilization and Appropriateness across the United States

Background

Substantial geographic variation exists in percutaneous coronary intervention (PCI) use across the United States. It is unclear the extent to which high PCI utilization can be explained by PCI for inappropriate indications. The objective of this study was to examine the relationship between PCI rates across regional healthcare markets utilizing hospital referral regions (HRRs) and PCI appropriateness.

Methods

The number of PCI procedures in each HRR was obtained from the 2010 100% Medicare limited data set. HRRs were divided into quintiles of PCI utilization with increasing rates of utilization progressing to quintile 5. NCDR CathPCI Registry^® data were used to evaluate patient characteristics, appropriate use criteria (AUC), and outcomes across the HRR quintiles defined by PCI utilization with the study population restricted to HRRs where ≥ 80% of the PCIs were performed at institutions participating in the registry. PCI appropriateness was defined using 2012 AUC by the American College of Cardiology (ACC)/American Heart Association (AHA)/The Society for Cardiovascular Angiography and Interventions (SCAI).

Results

Our study cohort comprised of 380,981 patients treated at 178 HRRs. Mean PCI rates per 1,000 increased from 4.6 in Quintile 1 to 10.8 in Quintile 5. The proportion of non-acute PCIs was 27.7% in Quintile 1 increasing to 30.7% in Quintile 5. Significant variation (p < 0.001) existed across the quintiles in the categorization of appropriateness across HRRs of utilization with more appropriate PCI in lower utilization areas (Appropriate: Q1, 76.53%, Q2, 75.326%, Q3, 75.23%, Q4, 73.95%, Q5, 72.768%; Inappropriate: Q1 3.92%, Q2 4.23%, Q3 4.32%, Q4 4.35%, Q5 4.05%; Uncertain: Q1 8.29%, Q2 8.84%, Q3 8.08%, Q4 9.01%, Q5 8.93%; Not Mappable: Q1 11.26%, Q2 11.67%, Q3 12.37%, Q4 12.69%, Q5 14.34%). There was no difference in risk-adjusted mortality across quintiles of PCI utilization.

Conclusions

Geographic regions with lower PCI rates have a higher proportion of PCIs performed for appropriate indications. Areas that perform more PCIs also appear to perform more elective PCI and many could not be mapped by the AUC.     

Variation in nuclear DNA content across environmental gradients: a quantile regression analysis

The nuclear DNA content of angiosperms varies by several orders of magnitude. Previous studies suggest that variation in 2C DNA content (i.e. the amount of DNA in G1 phase nuclei, also referred to as the 2C-value) is correlated with environmental factors, but there are conflicting reports in the literature concerning the nature of these relationships. We examined variation in 2C DNA content for 401 species in the ecologically diverse California flora in relation to the mean July maximum temperature, January minimum temperature, and annual precipitation within the geographical ranges of these species. Species with small 2C-values predominate in all environments. Species with large 2C-values occur at intermediate July maximum temperatures, and decline in frequency at both extremes of the July temperature gradient, and with decreasing annual precipitation. Our analysis demonstrates the utility of quantile regression for statistical inference of complex distributions such as these. The method supports our observation that relationships between nuclear DNA content and environmental factors are stronger for species with large 2C-values.     

Molecular identification and analysis of Borrelia burgdorferi sensu lato in lizards in the southeastern United States

Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.     

Non-native plant invasions of United States National Parks

The United States National Park Service was created to protect and make accessible to the public the nation’s most precious natural resources and cultural features for present and future generations. However, this heritage is threatened by the invasion of non-native plants, animals, and pathogens. To evaluate the scope of invasions, the USNPS has inventoried non-native plant species in the 216 parks that have significant natural resources, documenting the identity of non-native species. We investigated relationships among non-native plant species richness, the number of threatened and endangered plant species, native species richness, latitude, elevation, park area and park corridors and vectors. Parks with many threatened and endangered plants and high native plant species richness also had high non-native plant species richness. Non-native plant species richness was correlated with number of visitors and kilometers of backcountry trails and rivers. In addition, this work reveals patterns that can be further explored empirically to understand the underlying mechanisms.     

Sources of bacteria in outdoor air across cities in the midwestern United States

Bacteria are abundant in the atmosphere, where they often represent a major portion of the organic aerosols. Potential pathogens of plants and livestock are commonly dispersed through the atmosphere, and airborne bacteria can have important effects on human health as pathogens or triggers of allergic asthma and seasonal allergies. Despite their importance, the diversity and biogeography of airborne microorganisms remain poorly understood. We used high-throughput pyrosequencing to analyze bacterial communities present in the aerosol fraction containing fine particulate matter of ≤2.5 μm from 96 near-surface atmospheric samples collected from cities throughout the midwestern United States and found that the communities are surprisingly diverse and strongly affected by the season. We also directly compared the airborne communities to those found in hundreds of samples representing potential source environments. We show that, in addition to the more predictable sources (soils and leaf surfaces), fecal material, most likely dog feces, often represents an unexpected source of bacteria in the atmosphere at more urbanized locations during the winter. Airborne bacteria are clearly an important, but understudied, component of air quality that needs to be better integrated into efforts to measure and model pollutants in the atmosphere.     

Molecular Surveillance of Low Pathogenic Avian Influenza Viruses in Wild Birds across the United States: Inferences from the Hemagglutinin Gene

A United States interagency avian influenza surveillance plan was initiated in 2006 for early detection of highly pathogenic avian influenza viruses (HPAIV) in wild birds. The plan included a variety of wild bird sampling strategies including the testing of fecal samples from aquatic areas throughout the United States from April 2006 through December 2007. Although HPAIV was not detected through this surveillance effort we were able to obtain 759 fecal samples that were positive for low pathogenic avian influenza virus (LPAIV). We used 136 DNA sequences obtained from these samples along with samples from a public influenza sequence database for a phylogenetic assessment of hemagglutinin (HA) diversity in the United States. We analyzed sequences from all HA subtypes except H5, H7, H14 and H15 to examine genetic variation, exchange between Eurasia and North America, and geographic distribution of LPAIV in wild birds in the United States. This study confirms intercontinental exchange of some HA subtypes (including a newly documented H9 exchange event), as well as identifies subtypes that do not regularly experience intercontinental gene flow but have been circulating and evolving in North America for at least the past 20 years. These HA subtypes have high levels of genetic diversity with many lineages co-circulating within the wild birds of North America. The surveillance effort that provided these samples demonstrates that such efforts, albeit labor-intensive, provide important information about the ecology of LPAIV circulating in North America.     

Molecular characterization of the Trichomonas gallinae morphologic complex in the United States

Forty-two Trichomonas gallinae isolates were molecularly characterized to determine whether isolates differed in genetic sequence of multiple gene targets depending on host species or geographical location. The 5.8S ribosomal RNA (rRNA) and flanking internal transcribed spacer (ITS) gene regions were amplified by polymerase chain reaction, and the sequences were analyzed phylogenetically. The results of the sequence analysis strongly suggest at least 2 species may exist within the T. gallinae morphologic complex. Based on ITS sequences, one group demonstrated high nucleotide identity to the 3 T. gallinae sequences available in GenBank, whereas the second group was more closely related to T. vaginalis (98%) than to T. gallinae (92%). Two common ground-dove (Columbina passerina) isolates shared a 95% identity with T. vaginalis and a 92% identity with T. gallinae and T. tenax. Sequence analysis of both the 18S rRNA and alpha-tubulin genes from a subset of the isolates supports the 5.8S-ITS sequence results. All of the T. vaginalis-like isolates originated from Arizona, California, or Texas, whereas T. gallinae isolates were found in all sampled states. Both T. vaginalis-like and T. gallinae isolates were involved in trichomoniasis outbreaks in California and Arizona.