Protocol for sampling environmental sites for legionellae

A protocol for sampling environmental sites was developed and used to identify possible sources of Legionella species in support of epidemiologic investigations at two hospitals. In hospital A, legionellae were isolated from 43 of 106 (40%) different sites. Three separate Legionella pneumophila serotypes and a previously unrecognized species were present in different combinations in the positive samples. Two of five cooling towers contained the same L. pneumophila serogroup 1 monoclonal type (1,2,4,5) as was isolated from patients. The same monoclonal type was also isolated from make-up water for the two cooling towers, a hot water tank, water separators in four main air compressor systems for respiratory therapy, and cold and hot water faucets. In hospital B, 13 of 37 (38%) sample sites contained legionellae, all of which were L. pneumophila serogroup 1. The monoclonal type matching isolates from patients (1,2,4,5) was found at the highest concentration in a hot water tank, but it was also present at four other sample sites. Since legionellae not related to disease may be found in many of the sites sampled, an epidemiologic association with the probable source should be established before intervention methods, such as disinfection, are undertaken.     

Relative sensitivities of environmental legionellae to selective isolation procedures.

A survey of water samples to determine the efficacy of standard procedures for the isolation of environmental legionellae was conducted. Marked variations in intraspecies resistance to selective agents and treatments were observed, and in experiments with one of the isolates, the response was modified by culture conditions. Five selective procedures incorporating acid (pH 2.2) and heat (50 degrees C, 30 min) treatments, with and without plating on buffered charcoal-yeast extract agar supplemented with vancomycin (5 micrograms/ml), polymyxin B (60 U/ml), and cycloheximide (80 micrograms/ml), caused 5 to 99% decreases in viable counts of pure cultures in water suspensions. The differences in the responses of the cultures to the five treatments were statistically significant. Cells in retained samples of naturally contaminated water from which the original cultures had been isolated were significantly less sensitive than artificially grown isolates. The sensitivities of the laboratory-grown cells to the treatments were affected by the length of incubation on buffered charcoal-yeast extract agar. Whereas acid resistance increased after 24 h of incubation, resistance to the antibiotic mixture decreased.     

Relative sensitivities of environmental legionellae to selective isolation procedures

A survey of water samples to determine the efficacy of standard procedures for the isolation of environmental legionellae was conducted. Marked variations in intraspecies resistance to selective agents and treatments were observed, and in experiments with one of the isolates, the response was modified by culture conditions. Five selective procedures incorporating acid (pH 2.2) and heat (50 degrees C, 30 min) treatments, with and without plating on buffered charcoal-yeast extract agar supplemented with vancomycin (5 micrograms/ml), polymyxin B (60 U/ml), and cycloheximide (80 micrograms/ml), caused 5 to 99% decreases in viable counts of pure cultures in water suspensions. The differences in the responses of the cultures to the five treatments were statistically significant. Cells in retained samples of naturally contaminated water from which the original cultures had been isolated were significantly less sensitive than artificially grown isolates. The sensitivities of the laboratory-grown cells to the treatments were affected by the length of incubation on buffered charcoal-yeast extract agar. Whereas acid resistance increased after 24 h of incubation, resistance to the antibiotic mixture decreased.     

Zooplankton sampling strategies for environmental studies

This study characterizes sources of variation in total zooplankton abundance estimates at seven stations within the 5–10 m depth contour of southeastern Lake Michigan which were sampled monthly, April through October, for the 1975 to 1979 period. Month, year, and station were statistically significant factors affecting abundance estimates as were all interactions. Month was the largest source of variance either as a main effect or interaction. Smallest coefficients of variation were associated with subsampling (mean 6.1%) and replicate sampling (mean 15.1%). The between-station coefficient of variation averaged 39.0% and tended to be highest during the summer. For a given station and month, the between-year coefficient of variation averaged 73.4% while the between-month coefficient of variation for a single station in a given year averaged 95.1%. A table shows the estimated number of replications necessary to detect a true difference in two population means as a function of coefficient of variation. Environmental studies designed to detect spatial alterations should conduct such analyses on a cruise-by-cruise basis. Cruises should consist of a large number of stations and be conducted at least once during each season. Studies designed to detect temporal alterations require more frequent sampling because of the greater variability associated with temporal data sets. Because spatial variability adds little to the overall variability of such data sets, only a few representative stations need be sampled during each cruise.     

Importance sampling for the infinite sites model

Importance sampling or Markov Chain Monte Carlo sampling is required for state-of-the-art statistical analysis of population genetics data. The applicability of these sampling-based inference techniques depends crucially on the proposal distribution. In this paper, we discuss importance sampling for the infinite sites model. The infinite sites assumption is attractive because it constraints the number of possible genealogies, thereby allowing for the analysis of larger data sets. We recall the Griffiths-Tavaré and Stephens-Donnelly proposals and emphasize the relation between the latter proposal and exact sampling from the infinite alleles model. We also introduce a new proposal that takes knowledge of the ancestral state into account. The new proposal is derived from a new result on exact sampling from a single site. The methods are illustrated on simulated data sets and the data considered in Griffiths and Tavaré (1994).     

Risk factors for contamination of domestic hot water systems by legionellae.

To assess risk factors associated with the contamination of the domestic environment by legionellae, 211 houses in the Quebec City area were randomly selected and water samples were collected from the hot water tank, the shower heads, and the most frequently used faucet. After centrifugation, concentrated samples were seeded in triplicate on BCYE and GPV media. Data on the characteristics of the hot water system and plumbing in the house and on the personal habits of the occupants were collected for each house. Among these 211 houses, hot water was provided by either an oil or gas heater in 33 and by an electric heater in 178. Legionellae were isolated from none of the samples from houses with oil or gas heaters and from 39% (69 of 178) of those with electric water heaters (P less than 0.0001). This association remained highly significant after control for water temperature and other variables in a stratified analysis. In the 178 houses with an electric heater, 12% of the faucets, 15% of the shower heads, and 37% of the water heaters were contaminated. Legionella pneumophila serogroups 2 and 4 were the most frequently isolated strains. Logistic regression showed that factors associated with electric water heater contamination were (i) location of the house in older districts of the city (P less than 0.0001), (ii) old age of the water heater (P = 0.003), and (iii) low water temperature (P = 0.05). Contamination of the water heater was the only factor significantly associated with the contamination of peripheral outlets (P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of membrane filters for recovery of legionellae from water samples.

The procedure currently used for isolating legionellae from environmental samples recommend filtration through a 0.2-microns-pore-size polycarbonate filter. In this study we evaluated the performance of 23 other filters composed of various materials and having various pore sizes. We prefer the 0.2-micron-pore-size Gelman Supor filter because of its high level of recovery, faster filtration rate, and ease of handling.     

Protocol for efficient plasma sampling for low density lipoprotein turnover studies

Radiolabeled low density lipoprotein (LDL) is commonly used to study the turnover of LDL apolipoprotein B (apoB), the major protein component of LDL. Following an intravenous injection of radioiodinated LDL, typical sampling schedules have including 20-25 samples over a 14-day period with frequent sampling during the first 12 hr and daily samples thereafter. This is a burdensome task for subjects and investigators. To improve acceptance of the procedure, we have examined the effects of reduced sampling schedules upon the estimation of the fractional catabolic rate (FCR) for LDL apoB. Data from 36 different sets of LDL decay curves obtained from investigations of subjects with a variety of lipoprotein phenotypes have been used to test these schedules. Our results indicate that by choosing specific intervals over a 14-day period only 10 samples are sufficient to accurately determine the fractional catabolic rate for LDL in plasma. This reduced sampling schedule should facilitate the study of LDL turnover in large groups of subjects as outpatients.     

Risk factors for contamination of domestic hot water systems by legionellae.

To assess risk factors associated with the contamination of the domestic environment by legionellae, 211 houses in the Quebec City area were randomly selected and water samples were collected from the hot water tank, the shower heads, and the most frequently used faucet. After centrifugation, concentrated samples were seeded in triplicate on BCYE and GPV media. Data on the characteristics of the hot water system and plumbing in the house and on the personal habits of the occupants were collected for each house. Among these 211 houses, hot water was provided by either an oil or gas heater in 33 and by an electric heater in 178. Legionellae were isolated from none of the samples from houses with oil or gas heaters and from 39% (69 of 178) of those with electric water heaters (P less than 0.0001). This association remained highly significant after control for water temperature and other variables in a stratified analysis. In the 178 houses with an electric heater, 12% of the faucets, 15% of the shower heads, and 37% of the water heaters were contaminated. Legionella pneumophila serogroups 2 and 4 were the most frequently isolated strains. Logistic regression showed that factors associated with electric water heater contamination were (i) location of the house in older districts of the city (P less than 0.0001), (ii) old age of the water heater (P = 0.003), and (iii) low water temperature (P = 0.05). Contamination of the water heater was the only factor significantly associated with the contamination of peripheral outlets (P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance sampling for Lambda-coalescents in the infinitely many sites model

We present and discuss new importance sampling schemes for the approximate computation of the sample probability of observed genetic types in the infinitely many sites model from population genetics. More specifically, we extend the ‘classical framework’, where genealogies are assumed to be governed by Kingman’s coalescent, to the more general class of Lambda-coalescents and develop further Hobolth et al.’s (2008) idea of deriving importance sampling schemes based on ‘compressed genetrees’. The resulting schemes extend earlier work by Griffiths and Tavaré (1994), Stephens and Donnelly (2000), Birkner and Blath (2008) and Hobolth et al. (2008). We conclude with a performance comparison of classical and new schemes for Beta- and Kingman coalescents.     

Evaluation of sampling tools for environmental sampling of bacterial endospores from porous and nonporous surfaces

Aims: Having and executing a well-defined and validated sampling protocol is critical following a purposeful release of a biological agent for response and recovery activities, for clinical and epidemiological analysis and for forensic purposes. The objective of this study was to address the need for validated sampling and analysis methods called out by the General Accounting Office and others to systematically compare the collection efficiency of various swabs and wipes for collection of bacterial endospores from five different surfaces, both porous and nonporous. This study was also designed to test the collection and extraction solutions used for endospore recovery from swabs and wipes. Methods and Results: Eight collection tools, five swabs and three wipes, were used. Three collection/preservation solutions were evaluated: an ink jet aerosol generator was used to apply Bacillus subtilis endospores to five porous and nonporous surfaces. The collection efficiencies of the swabs and wipes were compared using a statistical multiple comparison analysis. Conclusions: The ScottPure^® wipe had the highest collection efficiency and phosphate-buffered saline (PBST) with 0·3% Tween was the best collection solution of those tested. Significance and Impact of the Study: Validated sampling for potential biological warfare is of significant importance and this study answered some relevant questions.     

Airborne environmental endotoxin: a cross-validation of sampling and analysis techniques.

A standard method for measurement of airborne environmental endotoxin was developed and field tested in a fiberglass insulation-manufacturing facility. This method involved sampling with a capillary-pore membrane filter, extraction in buffer using a sonication bath, and analysis by the kinetic-Limulus assay with resistant-parallel-line estimation (KLARE). Cross-validation of the extraction and assay method was performed by comparison with methanolysis of samples followed by 3-hydroxy fatty acid (3-OHFA) analysis by gas chromatography-mass spectrometry. Direct methanolysis of filter samples and methanolysis of buffer extracts of the filters yielded similar 3-OHFA content (P = 0.72); the average difference was 2.1%. Analysis of buffer extracts for endotoxin content by the KLARE method and by gas chromatography-mass spectrometry for 3-OHFA content produced similar results (P = 0.23); the average difference was 0.88%. The source of endotoxin was gram-negative bacteria growing in recycled washwater used to clean the insulation-manufacturing equipment. The endotoxin and bacteria become airborne during spray cleaning operations. The types of 3-OHFAs in bacteria cultured from the washwater, present in the washwater and in the air, were similar. Virtually all of the bacteria cultured from air and water were gram negative composed mostly of two species, Deleya aesta and Acinetobacter johnsonii. Airborne countable bacteria correlated well with endotoxin (r2 = 0.64). Replicate sampling showed that results with the standard sampling, extraction, and Limulus assay by the KLARE method were highly reproducible (95% confidence interval for endotoxin measurement +/- 0.28 log10). These results demonstrate the accuracy, precision, and sensitivity of the standard procedure proposed for airborne environmental endotoxin.     

Tracer mixing: sites of tracer infusion and sampling

Controversy exists in the literature concerning the correct infusion and sampling sites in studies measuring substrate turnover rates. To investigate this problem, we examined the results obtained with various infusion and sampling sites in 7 anesthetized dogs. [1-14C]lactate was infused by a primed continuous infusion method in three different sites (the left ventricle, ascending aorta, and the aortic arch) in a sequential fashion; samples were obtained simultaneously from five sites (femoral artery, carotid artery, pulmonary artery, superior vena cava and inferior vena cava) for each of the three different infusion sites. [U-13C]lactate was also infused in a femoral vein and simultaneous samples were obtained in the carotid artery and femoral artery for analysis of the stable isotope. [14C]lactate analysis demonstrated that infusion of the tracer into the left ventricular chamber resulted in a uniform distribution in the systemic circulation. Infusion into the ascending aorta near the aortic valve resulted in uniform distribution of tracer in four out of five experiments. Tracer infusion into the aortic arch resulted in nonuniform systemic distribution of tracer. The [U-13C]lactate results showed that infusion into the femoral vein gives uniform systemic distribution, similar to that observed with left ventricular infusion. The pulmonary artery lactate specific activities varied from those in the superior vena cava. Thus, this study shows that the tracer must be infused in the left ventricle or upstream from this chamber to obtain optimal systemic distribution. Vena caval sampling, especially superior vena caval sampling, will not give a consistent mixed venous concentration of the lactate tracer. Therefore, aortic tracer infusion with vena caval sampling may lead to errors in determining substrate turnover values.     

A note on the sampling theory for infinite alleles and infinite sites models

This note considers sampling theory for a selectively neutral locus where it is supposed that the data provide nucleotide sequences for the genes sampled. It thus anticipates that technical advances will soon provide data of this form in volume approaching that currently obtained from electrophoresis. The assumption made on the nature of the data will require us to use, in the terminology ofKimura (Theor. Pop. Biol.2, 174–208 (1971)), the “infinite sites” model of Karlin and McGregor (Proc. Fifth Berkeley Symp. Math. Statist. Prob.4, 415–438 (1967)) rather that the “infinite alleles” model of Kimura and Crow (Genetics49, 174–738 (1964)). We emphasize that these two models refer not to two different real-world circumstances, but rather to two different assumptions concerning our capacity to investigate the real world. We compare our results where appropriate with corresponding sampling theory of Ewens (Theor. Pop. Biol.3, 87–112 (1972)) for the “infinite alleles” model. Note finally that some of our results depend on an assumption of independence of behavior at individual sites; a parallel paper byWatterson (submitted for publication (1974)) assumes no recombination between sites. Real-world behavior will lie between these two assumptions, closer to the situation assumed by Watterson than in this note. Our analysis provides upper bounds for increased efficiency in using complete nucleotide sequences.     

Rethinking taxon sampling in the light of environmental sequencing

Environmental DNA sequencing efforts of substrates such as soil, wood, and seawater have been found to present very different views of the underlying biological communities compared with efforts based on morphological examination and culture studies. The taxonomic affiliation of many of these environmental sequences cannot be settled with certainty due to the lack of proximate reference sequences in the corpus of public sequence data, and they are typically submitted to the international sequence databases without much indication of their relatedness. The scientific community has proved reluctant to include such unnamed sequences in phylogenetic analyses and taxonomic studies, but the present study shows such a position to be not only largely unwarranted but also potentially unsound. The sequences of 48 published fungal alignments of the nuclear ribosomal internal transcribed spacer region were subjected to similarity searches in the sequence databases to recover environmental sequences with a clear bearing on the respective ingroup. An average of 20 environmental sequences were added to each alignment, and upon rerunning the phylogenetic analyses of each study we found that topological rearrangements involving the original ingroup sequences were observed for no less than 29 (60%) of the studies. In nearly 20% of these cases, the rearrangements were large enough to question or even overthrow at least one conclusion presented in the original studies. The basal branching order was similarly subject to changes in 16% of the applicable studies. Environmental sequences are thus not only relevant in ecological research but form a requisite source of information also in systematics and taxonomy.
© The Willi Hennig Society 2010.     

Importance of sites of tracer administration and blood sampling in relation to leucine metabolism. Practical considerations.

For the same infusion site of L-[1-14C]leucine, sampling downstream of arterial blood underestimates leucine turnover, whereas sampling of venous blood overestimates turnover. Further, the lungs release a small but consistent amount of leucine into the blood. Unlabelled leucine also is produced by the portal-drained viscera, and some is removed immediately by the liver. These sources of leucine should thus be considered in turnover calculations.     

小麦和玉米根系取样位置优化确定及根系分布模拟

为了确定小麦(Triticum aestivum)、玉米(Zea mays)根系的最优取样位置和更准确地模拟根长密度在土壤剖面的分布, 在冬小麦和夏玉米的灌浆后期, 采用根钻法取样, 比较了不同取样位置对根系分布的影响; 采用Gerwitz和Page模型对根长密度的分布进行了模拟。结果表明, 冬小麦行间和行上取样在0-10 cm土层根长密度差异显著, 在10 cm以下土层差异减少。在确定根长密度分布的取样中, 在0-20 cm土层应考虑根长密度分布的空间差异, 即行上密度大于行间密度; 而在20-100 cm土层, 需要考虑行间根长密度大于行上的空间差异; 在1 m以下土层两个位置的差异逐渐消失, 可不考虑空间差异。夏玉米根长密度在上层土壤表现出距离植株不同位置差异显著的特征。植株位置(株上)、距植株10 cm和距植株20 cm位置根长密度在土壤中的分布特征是: 0-10 cm土层3个位置根长密度差异在50%以上, 根长密度大小是株上>距植株10 cm>距植株20 cm; 而在10-30 cm层次, 根长密度表现为距植株10 cm>株上>距植株20 cm, 30-50 cm土层株上位置的根长密度最小, 50 cm以下各位置根长密度差异不明显。对于玉米根系取样, 50 cm以上土层需要考虑根长密度的空间差异, 50 cm以下土层可不考虑。采用Gerwitz和Page模型, 结合华北平原机械化耕作下形成的土壤犁底层变厚及其犁底层容重增加对根系分布的影响, 在模型中加入土壤容重参数订正可以使模型更准确地模拟根长密度在土壤剖面的分布。     

Distribution of Legionella longbeachae serogroup 1 and other legionellae in potting soils in Australia.

Legionella longbeachae serogroup 1 and other Legionella spp. were isolated from 73% of 45 potting soils made in Australia by 13 manufacturers but were not detected in 19 potting soils made in Greece, Switzerland, and the United Kingdom examined between March 1989 and May 1990. Several Legionella species were isolated from a small number of samples of uncomposted pine sawdusts, but it is not known whether sawdust was the source of some of the legionellae found in potting soils. Legionella spp. persisted for periods ranging from 3 to 10 months in a potting soil held at temperatures between -20 and 35 degrees C. Isolates of L. longbeachae serogroup 1 from soil did not grow at 43 degrees C, a temperature which was also lethal for this species in soil. Most Legionella spp. isolated from potting and natural soils belonged to one distinct group according to analysis of ubiquinones and were serologically related to several known species in this group. A small number of potting soils contained L. pneumophila and L. micdadei.     

Relationships between environmental variables and vegetation across mountain wetland sites,N. Iran

The mountain wetlands studied represent a unique habitat on the southern slopes of the Alborz mountain range, the second largest range in Iran. In comparison with other parts of this range the western section is ecologically and botanically unknown. Floristic and vegetation variation were assessed using diverse environmental variables along a broad altitudinal span (350 m to 3200 m a.s.l.). Using both statistical and ordination analyses floristic variation was assessed on three defined altitudinal belts which were delimited based on Alborz macro-climatic boundaries. The distribution of individual wetland plant species, of phytogeographic elements and of life-forms all differ among altitudinal belts. This result is also shown in both direct and indirect analyses of ordinations. The proportion of geophytes significantly increases with altitude and geophytes are very well represented in the upper altitudinal belt. The number of species of a narrow phytogeographical distribution (e.g. endemics) increases with altitude, soil pH and EC declined with altitude. The first axis of DCA ordination with passively projected environmental variables indicates that, organic matter and concentration of Fe²⁺ are increased toward higher altitude. The second axis of ordination is related to both soil texture and slope inclination. The distribution of species in the CCA species plot is also close to the distribution of those in the DCA ordination. This study indicates that altitude and slope together with other dependent environmental variables (pH, EC, Ca²⁺ and soil texture) are the main ecological factors controlling species distribution across the Western Alborz wetland sites.