Inter‐laboratory validation of a rapid assay for the detection and quantification of Legionella spp. in water samples

Aims: To compare the standard culture method with a new, rapid test (ScanVIT‐Legionella?) using fluorescently labelled gene probes for the detection and enumeration of Legionella spp. The new technique was validated through experiments conducted on both artificially and naturally contaminated water and through an inter‐laboratory comparison. Methods and Results: All samples were processed by the ScanVIT test according to the manufacturer’s instructions and by a culture method (ISO 11731). ScanVIT detected significantly more positive samples, although concentrations were similar and a strong positive correlation between the two methods was observed (r = 0·888, P < 0·001). The new test was more accurate in identifying the co‐presence of Legionella pneumophila and Leg. non‐pneumophila. ScanVIT showed a slightly higher Legionella recovery from water samples artificially contaminated with Leg. pneumophila alone or together with Pseudomonas aeruginosa. Lastly, the inter‐laboratory comparison revealed that the ScanVIT test exhibits a lower variability than the traditional culture test (mean coefficient of variation 8·7 vs 16·1%). Conclusions: The results confirmed that the ScanVIT largely overlaps the reference method and offers advantages in terms of sensitivity, quantitative reliability and reduced assay time. Significance and Impact of the Study: The proposed method may represent a useful validated alternative to traditional culture for the rapid detection and quantification of Legionella spp. in water.     

Evaluation of real‐time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms

Aims: To assess two real‐time PCR methods (the Riviere and Qvarnstrom assays) for environmental Acanthamoeba. Methods and Results: DNA extracted from Acanthamoeba castellanii taken from water and biofilms of cooling towers was analysed by the Riviere and Qvarnstrom assays. To quantify environmental Acanthamoeba, the calibration curves (DNA quantity vs cell number) were constructed with samples spiked with A. castellanii. The calibration curves for both quantitative PCR assays showed low variation (coefficient of variation of C_t≤ 5·7%) and high linearity (R² ≥ 0·99) over six orders of magnitudes with detection limit of three cells per water sample. DNA quantity determined by Qvarnstrom assay was equivalent between trophozoites and cysts (P = 0·49), whereas a significant difference was observed with Riviere assay (P < 0·0001). Riviere assay failed to detect Acanthamoeba in 21% (15/71) of the environmental samples which were positively detected by Qvarnstrom assay, while one sample (1·4%) was shown positive by Riviere assay but negative by Qvarnstrom assay. Moreover, Acanthamoeba counts by Qvarnstrom assay were greater than those by Riviere assay (P < 0·0001). Conclusions: Qvarnstrom assay performs better than Riviere assay for detection and quantification of Acanthamoeba in anthropogenic water and biofilms. Significance and Impact of the Study: Qvarnstrom assay may significantly contribute to a better knowledge about the distribution and abundance of Acanthamoeba in environments.     

Longitudinal evaluation of the efficacy of heat treatment procedures against Legionella spp. in hospital water systems by using a flow cytometric assay

Legionella spp. are frequently isolated in hospital water systems. Heat shock (30 min at 70°C) is recommended by the World Health Organization to control its multiplication. The aim of the study was to evaluate retrospectively the efficacy of heat treatments by using a flow cytometry assay (FCA) able to identify viable but nonculturable (VBNC) cells. The study included Legionella strains (L. pneumophila [3 clusters] and L. anisa [1 cluster]) isolated from four hot water circuits of different hospital buildings in Saint-Etienne, France, during a 20-year prospective surveillance. The strains recovered from the different circuits were not epidemiologically related, but the strains isolated within a same circuit over time exhibited an identical genotypic profile. After an in vitro treatment of 30 min at 70°C, the mean percentage of viable cells and VBNC cells varied from 4.6% to 71.7%. The in vitro differences in heat sensitivity were in agreement with the observed efficacy of preventive and corrective heating measures used to control water contamination. These results suggest that Legionella strains can become heat resistant after heating treatments for a long time and that flow cytometry could be helpful to check the efficacy of heat treatments on Legionella spp. and to optimize the decontamination processes applied to water systems for the control of Legionella proliferation.     

Covalently linked immunomagnetic separation/adenosine triphosphate technique (Cov‐IMS/ATP) enables rapid,in‐field detection and quantification of Escherichia coli and Enterococcus spp. in freshwater and marine environments

Aims: Developing a rapid method for detection of faecal pollution is among the critical goals set forth by the Environmental Protection Agency in its revision of water quality criteria. The purpose of this study is to devise and test covalently linked antibody–bead complexes for faecal indicator bacteria (FIB), specifically Escherichia coli or Enterococcus spp., in measuring water quality in freshwater and marine systems. Methods and Results: Covalently linked complexes were 58–89% more robust than antibody–bead complexes used in previous studies. Freshwater and marine water samples analysed using covalently linked immunomagnetic separation/adenosine triphosphate quantification technique (Cov‐IMS/ATP) and culture‐based methods yielded good correlations for E. coli (R = 0·87) and Enterococcus spp. (R = 0·94), with method detection limits below EPA recreational water quality health standards for single standard exceedances (E. coli– 38 cells per 100 ml; Enterococcus spp. – 25 cells per 100 ml). Cov‐IMS/ATP correctly classified 87% of E. coli and 94% of Enterococcus spp. samples based on these water quality standards. Cov‐IMS/ATP was also used as a field method to rapidly distinguish differential loading of E. coli between two stream channels to their confluence. Conclusions: Cov‐IMS/ATP is a robust, in‐field detection method for determining water quality of both fresh and marine water systems as well as differential loading of FIB from two converging channels. Significance and Impact of the Study: To our knowledge, this is the first work to present a viable rapid, in‐field assay for measuring FIB concentrations in marine water environments. Cov‐IMS/ATP is a potential alternative detection method, particularly in areas with limited laboratory support and resources, because of its increased economy and portability.     

Comparisons of soil‐water content between a Moso bamboo (Phyllostachys pubescens) forest and an evergreen broadleaved forest in western Japan

In Japan, forests of Moso bamboo (Phyllostachys pubescens, an exotic invasive giant bamboo) have naturalized and expanded rapidly, replacing surrounding broadleaved and coniferous forests. To evaluate impacts caused by these forest‐type replacements on the hydrological cycle, soil‐water content and its spatial variability in a Moso bamboo forest were compared with those in an adjacent evergreen broadleaved forest, in a case study of a stand in western Japan (northern Kyushu). The volumetric soil‐water content averaged over depths between 0 and 60 cm was consistently higher in the bamboo stand than that in the broadleaved stand. These results contrast with previous studies comparing the soil‐water content in Moso bamboo forests with that in other forest types. The sum of canopy transpiration and soil evaporation (E) in the bamboo stand tended to be larger than that in the broadleaved stand. Small canopy interception loss was reported in the bamboo forest. Therefore, the large amount of E would counterbalance the small canopy interception loss in the bamboo forest. Differences in soil characteristics between the two stands may be the main factor causing differences in soil‐water content. Spatial variation in soil‐water content in the bamboo stand was larger than that in the broadleaved stand, confirming findings in a previous series of our study. This could happen because the well‐developed root‐system in the bamboo forest enhances preferential flow in the soil. To evaluate the effects of aggressive invasion of alien giant bamboo on the ecosystem functions, we recommend further studies measuring various hydrological components in various Moso bamboo forests.     

Tahibacter aquaticus gen. nov., sp. nov., a new gammaproteobacterium isolated from the drinking water supply system of Budapest (Hungary)

Three Gram-stain negative, aerobic, non-motile, non-spore-forming, rod-shaped bacterial strains, PYM5-11^T, RaM5-2 and PYM5-8, were isolated from the drinking water supply system of Budapest (Hungary) and their taxonomic positions were investigated by a polyphasic approach. All three strains grew optimally at 20-28 °C and pH 5-7 without NaCl. The G+C content of the DNA of the type strain was 65.4 mol%. On the basis of 16S rRNA gene sequence analysis, the isolates showed 94.5-94.9% sequence similarity to the type strain of Dokdonella koreensis and a similarity of 93.0-94.1% to the species of the genera Aquimonas and Arenimonas. The major isoprenoid quinone of the strains was ubiquinone Q-8. The predominant fatty acids were iso-C_15:0, iso-C_17:1ω9c, C_16:1ω7c, and C_16:0. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine, as well as several unidentified aminolipids and phospholipids were present. The 16S rRNA gene sequence analysis, the predominant fatty acids, the polar lipid composition, RiboPrint patterns, physiological and biochemical characteristics showed that the three strains were related but distinct from the type strains of the four recognized species of the genus Dokdonella, and indicated that the strains represented a new genus within the Gammaproteobacteria. The strain PYM5-11 (=DSM 21667^T=NCAIM B 02337^T) is proposed as the type strain of a new genus and species, designated as Tahibacter aquaticus gen. nov., sp. nov.     

Osmotic water permeability of plasma and vacuolar membranes in protoplasts I. High osmotic water permeability in radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>) root cells as measured by a new method

Intra- and transcellular water movements in plants are regulated by the water permeability of the plasma membrane (PM) and vacuolar membrane (VM) in plant cells. In the present study, we investigated the osmotic water permeability of both PM (P ( f1)) and VM (P ( f2)), as well as the bulk osmotic water permeability of a protoplast (P ( f(bulk))) isolated from radish (Raphanus sativus) roots. The values of P ( f(bulk)) and P ( f2) were determined from the swelling/shrinking rate of protoplasts and isolated vacuoles under hypo- or hypertonic conditions. In order to minimize the effect of unstirred layer, we monitored dropping or rising protoplasts (vacuoles) in sorbitol solutions as they swelled or shrunk. P ( f1) was calculated from P ( f(bulk)) and P ( f2) by using the 'three-compartment model', which describes the theoretical relationship between P ( f1), P ( f2) and P ( f(bulk)) (Kuwagata and Murai-Hatano in J Plant Res, 2007). The time-dependent changes in the volume of protoplasts and isolated vacuoles fitted well to the theoretical curves, and solute permeation of PM and VM was able to be neglected for measuring the osmotic water permeability. High osmotic water permeability of more than 500 mum s(-1), indicating high activity of aquaporins (water channels), was observed in both PM and VM in radish root cells. This method has the advantage that P ( f1) and P ( f2) can be measured accurately in individual higher plant cells.     

<Emphasis Type="Italic">Encentrum (Parencentrum) walterkostei</Emphasis> n. sp., a new dicranophorid rotifer (Rotatoria: Monogononta) from the high alpine zone of the Central Alps (Austria)

Leaf litter processing rates and fungal biomass on leaf detritus were compared in four streams of different water chemistry. The streams drained catchments underlain by different bedrock types and varied in mean pH from 4.3 to 7.5 and in mean alkalinity from 0.0 to 35.8 mg CaCO₃ l^–1. Processing rates were fastest in WS3 and WS4, which had a pH of 6.0; slowest in SFR, which had a pH of 4.3; and intermediate in HSR which had a pH of 7.5. Fungal biomass as measured by the fungal sterol, ergosterol, was similar in WS3, WS4, and HSR but was much lower in SFR. These results suggest that reduced processing rates in SFR were associated in part with reduced fungal biomass on the leaves, whereas reduced processing rates in HSR were not related to differences in fungal biomass on the leaves.The Unit is jointly sponsored by the U.S. Fish and Wildlife Service, the West Virginia Division of Natural Resources, West Virginia University, and the Wildlife Management Institute.     

Comparison of heart rate monitoring combined with indirect calorimetry and the doubly labelled water (2H218O) method for the measurement of energy expenditure in children

The aim of the present study was to compare data on 24-h energy expenditure (EE24h) in nine boys and ten girls (mean age 9.3 and 8.1 years, respectively) by heart rates (fc) combined with energy expenditure obtained from a 1-day stay in an indirect calorimeter (EEcal) and a 2-week period of normal living using the doubly labelled water method (EEdlw). Individual calibration curves were derived from fc and oxygen uptake measured during sleep (in the calorimeter), standing and walking on a treadmill. An estimation of energy expenditure based on 24-h fc monitoring (EEfc) was made during the stay in the calorimeter and on a normal school-day. Mean results showed an overestimation in EEfc compared to EEcal and EEdlw of 10.4% and 12.3% respectively, varying from 6.3% to 16.2%. These results confirmed earlier observations in adults that for a group the fc method overestimates EE24h by about 10%.     

A new method for in-situ monitoring of the underground development of Orobanche cumana in sunflower (Helianthus annuus) with a mini-rhizotron

AIMS: To develop an in-situ, non-destructive method for observation and monitoring of the underground developmental stages of the root parasite Orobanche cumana. SCOPE: The parasitic weed Orobanche causes severe damage to vegetables and field crops. Most of the damage caused to the crops occurs during the underground, unobservable parasitism stage. Sunflower (Helianthus annuus 'Adi') plants were planted in soil that was artificially inoculated with O. cumana seeds. Clear Plexiglas mini-rhizotron plastic observation tubes were inserted into the soil. Seed germination, early stage of penetration, and formation of tubercles and spikes were observed non-destructively and were monitored throughout the growing season by mean of a mini-rhizotron camera. Use of this technology enabled the complete individual parasite life cycle from the very early development (including germination) to Orobanche shoot to be monitored. In addition, the effect of the systemic herbicide Cadre (imazapic) on the development of O. cumana was inspected and quantified. CONCLUSIONS: This novel methodology facilitates the in-situ study of major aspects of the host-parasite interaction and of parasite suppression, such as parasitism dynamics, parasite growth rate, and the effect of chemical treatments on the parasite.     

Changes in distribution and abundance of the endangered damselfly <Emphasis Type=Italic>Mortonagrion hirosei</Emphasis> Asahina (Zygoptera: Coenagrionidae) in a reed community artificially established for its conservation

Population trends of the brackish water damselfly, Mortonagrion hirosei were studied for 4 years in the reed community artificially established for conservation of this endangered species. Because of difficulty with mark-and-recapture experiments on this small damselfly with weak wings in the large dense reed community, census counts using the line transect method were performed to estimate the population parameters. The reed rhizomes were transplanted in January of 2003. A few adults immigrated to the community in the flying season of this year, but they were restricted near the original habitat. The number of adults throughout the flying season was estimated at 1,000. In 2004, the population included both the immigrants from the original habitat and the emergences from the established habitat, and the total population was estimated at 10,000, and the daily density in peak flight season was 20% that in the original habitat. An estimated 23,000 individuals were found all over the established habitat in 2005. In 2006, the estimated number of adults in the established habitat was 45,600, and the population density increased almost equal to that in the original habitat. Therefore we can conclude that the damselfly had settled in the established habitat.     

Tricalcaria Han gen. nov., a remarkable new genus with three hind‐tibial spurs belonging to the tribe Trichopterygini,with description of a new species from China (Lepidoptera: Geometridae: Larentiinae)

Tricalcaria Han gen. nov. and its type species Tricalcaria stueningi Han sp. nov. are described from China. Morphological characters, including those of the male and female genitalia, are figured. The main diagnostic characters of Tricalcaria are: hind tibia with three spurs in both male and female; ventral margin of costal protrusion bearing a series of long, hook‐like, sclerotized setae in the male genitalia; female papilla analis modified and bearing over part of its surface broad blade‐like sclerotized setae. The wing pattern, venation, and genitalia of this genus are compared with those of the most closely related genera, Tristeirometa Holloway, 1997, Hypocometa Warren, 1896, and Phthonoloba Warren, 1893. Tribal placement is discussed, with the conclusion that the new genus should be placed in the tribe Trichopterygini.     

Evaluation of Two Triple‐Therapy Regimens with Metronidazole or Clarithromycin for the Eradication of H. pylori Infection in Vietnamese Children: a Randomized,Double‐Blind Clinical Trial

Background: Eradication of Helicobacter pylori infection in children in developing countries needs further investigations upon which to base treatment recommendations. The aim of the study was to compare two 2‐week triple therapies in a randomized double‐blind trial. Materials and Methods: In order not to exceed recommended dosages, the 238 H. pylori‐infected children, aged 3 to 15 years (mean 8.6), were divided in two weight categories receiving at weights 13–22 kg: lansoprazole 15 mg once‐daily and amoxicillin 500 mg twice‐daily with metronidazole 250 mg twice‐daily or clarithromycin 250 mg once‐daily; at weights 23–45 kg: lansoprazole 15 mg and amoxicillin 750 mg with metronidazole 500 mg or clarithromycin 250 mg, all administered twice daily. H. pylori status was assessed by culture and a monoclonal‐based antigen‐in‐stool test (Premier Platinum HpSA PLUS) and side effects by structured questionnaires. Results: The overall per‐protocol eradication (n = 233) was similar in the two treatment regimens, 62.1% for the metronidazole and 54.7% for the clarithomycin‐containing therapy. Eradication rate was higher in children ≥ 23 kg (70.9%) than in children < 23 kg (45.7%). In children ≥ 23 kg (n = 117) that received twice‐daily administration of all drugs, efficacy of the methronidazole and clarithromycin‐containing treatments were 69.5% and 72.4%, respectively. Conclusions: The two treatments gave similar eradication rates. Significant differences for both treatments were found by weight, which could be the result of the once‐daily proton pump inhibitor and clarithromycin and/or more antibiotic resistant strains in younger children.     

Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrid) plants

For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based “positive” selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 μM 2,4-D, 20 g l⁻¹ sucrose and 3 g l⁻¹ mannose. Plant regeneration was obtained on MS basal medium with 2.5 μM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 μM IBA, 20 g l⁻¹ sucrose, and 1.5 g l⁻¹ mannose. An increase in mannose concentration from permissive (1.5 g l⁻¹) to selective (3 g l⁻¹) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.