A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims:  Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies.
Method:  A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples.
Conclusions:  The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not.
Significance and Impact of the Study:  The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.     

Potential pathogenic bacteria in metalworking fluids and aerosols from a machining facility

The metalworking and machining industry utilizes recirculating metalworking fluids for integral aspects of the fabrication process. Despite the use of biocides, these fluids sustain substantial biological growth. Subsequently, the high-shear forces incurred during metalworking processing aerosolize bacterial cells and may cause dermatologic and respiratory effects in exposed workers. We quantified and identified the bacterial load for metalworking fluid and aerosol samples of a machining facility in the US Midwest during two seasons. To investigate the presence of potentially pathogenic bacteria in fluid and air, we performed 16S rRNA gene surveys. The concentration of total bacterial cells (including culturable and nonculturable cells) was relatively constant throughout the study, averaging 5.1 × 10? cells mL?1 in the fluids and 4.8 × 10? cells m?3 in the aerosols. We observed bacteria of potential epidemiologic significance from several different bacterial phyla in both fluids and aerosols. Most notably, Alcaligenes faecalis was identified through both direct sequencing and culturing in every sample collected. Elucidating the bacterial community with gene surveys showed that metalworking fluids were the source of the aerosolized bacteria in this facility.     

Characteristic Stimulation by Ethylene of Respiration in Dutch Iris Bulbs

The low steady-state basic respiration of iris bulbs (Iris bollandica cv.‘Wedgwood') at 30°C can be enhanced by ethylene. When ethylene is administered continuously a lag period of 4 hours is followed by an increase in the rate of respiration showing two peaks, one after 24 hours and the other after 3 to 4 days. Thereafter, the respiration rate decreases gradually, notwithstanding the presence of ethylene. Concentrations of less than 0.05 microlitre ethylene per litre have no effect, and the maximum effect is observed at concentrations of 3 microlitre per litre and higher. Between 0.2 and 2 microlitre ethylene per litre the peak values of the first maximum are linearly proportional to the logarithm of the ethylene concentration applied. The characteristics of the respiratory response in iris bulbs are probably similar to those described for potato tubers and non-climacteric fruits.     

Compliance of chest wall in obese subjects

Whereas studies in awake subjects have demonstrated that chest wall compliance (Ccw) is low in obese subjects, the one study performed on paralyzed obese subject found Ccw to be normal. The purpose of this study was to measure Ccw in awake obese subjects with the pulse-flow technique, a method which appears to detect respiratory muscle relaxation. Seven normal males, 14 obese males, and 8 obese females [body mass index (BMI) varied from 20 to 83 kg/m2] were studied in the seated position. Ccw was measured by blowing air at a constant flow into the mouth and lungs for approximately 2 s and calculated by dividing airflow in liters per second by the change in esophageal minus body surface pressure in centimeters of water per second. In normal and obese subjects we found no correlation between BMI and Ccw. We conclude that obesity does not decrease Ccw.     

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.     

不同种源麻栎生长性状的地理变异

本研究在江西永丰、浙江开化和安徽滁州3个试验点,以32个种源的麻栎试验林为对象,分析了不同种源间林木生长变异、主要经济性状(地上单株生物量)随林龄的动态变化,并基于AMMI模型进行生长性状稳定性分析和优良种源选择。结果表明: 3个试验点麻栎不同种源间的树高、胸(地)径和地上单株生物量均具有显著差异。麻栎地上单株生物量受地点、种源、种源×地点交互作用的显著影响,其中地点对生长变异影响最大,其次为种源和种源×地点。不同地点麻栎苗期(1～3年生)和幼林期(4～11年生)优良种源的选择结果具有较大差异。根据第11年地上单株生物量分别筛选了在当地表现较好的种源,江西永丰试验点7个优良种源,高出试验点均值15.6%～57.8%;浙江开化试验点7个优良种源,高出试验点均值19.2%～45.2%;安徽滁州试验点8个优良种源,高出试验点均值24.9%～63.3%。综合生长量和稳定性表现,筛选出4个适于3个地理区域短轮伐期炭用林培育的优良种源,这些种源地上单株生物量均值为36.55 kg,稳定性参数均值为0.97。     

Effects of stocking density and food density on survival,growth and yield of laboratory-reared larvae of sea bream Archosargus rhomboidalis (L.) (Sparidae)*

Growth and survival of sea bream, Archosargus rhomboidalis (L.) larvae were affected by both abundance of eggs that were initially stocked in 75 1 rearing systems and by the concentration of copepod nauplii and copepodites that were fed to larvae. Stocking levels were 2, 4, 8,16 or 32 per litre while food abundance was maintained at approximately 100, 500, 1500 or 3000 per litre. Experiments were of 16 days duration at 26° C. Survival was best, often exceeding 60%, when food levels were 1500 or 3000 per litre and when stocking density did not exceed 8 eggs per litre. Growth was best at the lowest stocking densities and highest food levels. The highest total yields in wet weight occurred at 8 per litre stock density and 3000 per litre food level. Mean wet weight per survivor and yield per stocked egg were greatest at the lowest stocking densities and highest food levels. A 500 per litre food level was marginal for growth and survival, and 100 per litre produced significant survival only at the 2 per litre stocking density. Two experiments at 6000 and 10 000 per litre food levels at 4 per litre stock density gave the best observed growth, and survival as good as in any other experiments. Possible relations between sea bream larvae and their food supply in the natural environment are discussed. Results also are discussed in terms of their possible contribution to aquaculture efforts.     

Microbiological Evaluation of a Large-Volume Air Incinerator

Two semiportable metal air incinerators, each with a capacity of 1,000 to 2,200 standard ft(3) of air per min, were constructed to sterilize infectious aerosols created for investigative work in a microbiological laboratory. Each unit has about the same air-handling capacity as a conventional air incinerator with a brick stack but costs only about one-third as much. The units are unique in that the burner housing and combustion chamber are air-tight and utilize a portion of the contaminated air stream to support combustion of fuel oil. Operation is continuous. Aerosols of liquid and dry suspensions of Bacillus subtilis var. niger spores and dry vegetative cells of Serratia marcescens were disseminated into the two incinerators to determine the conditions required for sterilization of contaminated air. With the latter organisms (concentration 2.03 x 10(7) cells/ft(3) of air), a temperature of 525 F (274 C), measured at the firebox in front of the heat exchanger, was sufficient for sterilization. To sterilize 1.74 x 10(7) and 1.74 x 10(9) wet spores of B. subtilis per ft(3), the required temperature ranged from 525 to 675 F (274 to 357 C) and 625 to 700 F (329 to 371 C), respectively. Air-sterilization temperature varied with each incinerator. This was because of innate differences of fabrication, different spore concentrations, and use of one or two burners With dry B. subtilis spores (1.86 x 10(8)/ft(3)), a temperature of 700 F was required for sterilization. With dry spores, no difference was noted in the sterilization temperature for the two incinerators.     

Measurement of cortisol in blow samples collected from free-swimming beluga whales (Delphinapterus leucas)

Blow or respiratory vapor collection is a noninvasive technique for monitoring the physiology of cetaceans and although this technique shows promise, a major challenge of blow collection is difficulty quantifying samples due to variable amounts of seawater contamination. Here, we aimed to (1) determine whether blow samples could be collected from free-swimming belugas, (2) assess whether urea could be used as a dilution marker to normalize blow samples, and (3) determine which factors influenced cortisol and urea concentrations. We collected a total of 252 blow samples from free-swimming belugas in the Churchill River in Manitoba, Canada. Cortisol, an indicator of individual stress and health, was detected in 65.87% of samples with concentrations ranging from 6.73 to 963.17 pg/ml of extract volume, while urea was detected in 90.48% of samples with concentrations ranging from 0.21 to 20.30 mg/L. We were unable to verify the efficacy of urea as a dilution marker to normalize blow samples from free-swimming belugas; however, absolute cortisol concentrations varied based on sample device and quantity rating. Although we demonstrated that blow can be successfully collected from free-swimming belugas, further refinement of this technique is needed before it can be used as a reliable method for physiological assessments.     

Rapid Detection of Small Numbers of Airborne Bacteria by a Membrane Filter Fluorescent-Antibody Technique

Bacteria from stirred settling aerosols were recovered and identified by a rapid fluorescent-antibody technique previously described by Danielsson. Calibrated air samples from Serratia marcescens aerosols were drawn through a Millipore aerosol filter holder fitted out with a nonfluorescent membrane filter (type HAB 047). With the aid of a high-power incident-light ultraviolet microscope, the individual bacterial cells, trapped on the filter, could be inspected after staining with appropriate conjugate on the filter surface.     

Morphology and formation of the eggshell in the tarnished plant bug, Lygus lineloaris (Palisot de Beauvois) (Hemiptera: Miridae)

Scanning and transmission electron microscopy were used to study the morphology and formation of the eggshell in the tarnished plant bug, Lygus lineolaris. Eggs are bean-shaped, with an operculum at the anterior end surrounded by a row of 36-40 respiratory horns. Three micropylar openings are on the operculum, and are sealed in oviposited eggs. The chorion consists of the chorion proper and the innermost chorionic layer. An air layer composed of colonnades is present in the chorion. The innermost chorionic layer is homogeneous and electron lucent. The follicle cells secrete electron dense materials that later coalesced into the reticulated vitelline membrane. This is followed by the deposition of the innermost chorionic layer by the follicle cells. After the primordial innermost chorionic layer is formed, follicle cells at the anterior pole of the oocyte secrete the scaffold for the colonnades in the air layer. Later, the primordial scaffold matrix is redistributed and localized at the lateral and posterior end of the oocyte where it becomes secondarily modified. At the end of choriogenesis, follicle cells at the anterior pole secrete the operculum and respiratory horns.     

Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification.

A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described. Bowes-melanoma cells were maintained in continuous serum-free culture. The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements. t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day. Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks. t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration. The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg.     

Responses of bulbar respiratory neurons to stimulation of receptive fields in the air passages

Extracellular recordings were made of changes in the firing pattern of 74 respiratory neurons in 23 cats anesthetized with Nembutal evoked by blowing atmospheric air into the nose or through an isolated segment of trachea. Respiratory unit (RU) responses were compared with accompanying changes in the activity of inspiratory and expiratory neuromotor units (NMUs) and the intratracheal pressure. These procedures were accompanied by changes in the frequency, depth, and rhythm of respiration and RU and NMU activity was activated or inhibited; RUs of all types responded to these stimuli. Responding RUs were found in various structures of the medullary respiratory center. Most RUs responded differently to stimulation of the air passages and inflation of the lungs. It is concluded that afferent impulses from the nose and trachea spread to all groups of bulbar RUs responsible for generating respiratory movements. This wide extent of the afferent projections of the air passages in structures of the respiratory center could play an important role both in defensive respiratory responses and in the regulation of eupnea.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 3, No. 6, pp. 620–630, November–December, 1971.     

利用角形几何特征和计算机视觉技术确定岩羊年龄的新方法

确定野生动物活体年龄是动物种群生态学研究的一项重要基础工作,然而,迄今为止,没有一种传统方法能高效精准地完成这项任务。岩羊角形态复杂且终身生长,其角形随年龄增长和观察角度变化极大。野外条件下,高效获取岩羊个体年龄信息的唯一方法就是拍摄其角的清晰照片。本研究旨在提出一种利用角形几何特征和计算机视觉技术确定野生岩羊活体年龄的新方法,其原理和操作如下:首先建立覆盖全部年龄段(1.5～15.5岁)的已知年龄羊角实体标本的3D模型(左、右角各22个);再在R程序控制下使其在3D空间中旋转,每旋转1°输出一张角形图片;最后计算每张角形输出图片与待定年龄角照片的形状相似性,找出相似性最高的角形图片对应的3D模型,该3D模型的年龄即为待定年龄羊角的实际年龄。30张已知年龄羊角照片的实验验证结果表明,上述年龄鉴定方法能高效地获得精准结果(准确率达100%)。     

Improved Large-Volume Sampler for the Collection of Bacterial Cells from Aerosol

A modified large-volume sampler was demonstrated to be an efficient device for the collection of mono-disperse aerosols of rhodamine B and poly-disperse aerosols of bacterial cells. Absolute efficiency for collection of rhodamine B varied from 100% with 5-μm particles to about 70% with 0.5-μm particles. The sampler concentrated the particles from 950 liters of air into a flow of between 1 and 2 ml of collecting fluid per min. Spores of Bacillus subtilis var. niger were collected at an efficiency of about 82% compared to the collection in the standard AGI-30 sampler. In the most desirable collecting fluids tested, aerosolized cells of Serratia marcescens, Escherichia coli, and Aerobacter aerogenes were collected at comparative efficiencies of approximately 90, 80, and 90%, respectively. The modified sampler has practical application in the study of aerosol transmission of respiratory pathogens.     

Measurement of Respiratory Volume for Virus Retention Studies in Mice

A pressure plethysmograph for measuring respiratory volume in mice during exposure to virus aerosols is described. The respiratory frequency and tidal volume were measured, and from these data the minute ventilation was calculated. The mean respiratory frequency of adult, male mice was 255 per min; the mean tidal volume of 0.18 ml was inversely related to respiratory frequency. The standardized mean minute ventilation rate was 1.46 ml per g of body weight. The respiratory frequency and tidal volume of CD-1 and HA/ICR strains of mice of the same age were similar. The respiratory retention rate for a 2.7-mum aerosol of vesicular stomatitis virus was 41%, and 58% of the virus retained was found in the trachea and lung.     

The veterinary management of a laryngeal air sac infection in a free-ranging mountain gorilla.

A purulent laryngeal air sac infection was diagnosed in a free-ranging adult male mountain gorilla (Gorilla gorilla beringei) in Central Africa. Successful treatment included the administration of antibiotics via blow dart and surgical drainage in the field. This case occurred during an outbreak of respiratory disease of undetermined etiology in this and other gorillas in the population.     

Assessing the short-term variability of bacterial composition in background aerosols of the Eastern Mediterranean during a rapid change of meteorological conditions

This study used a PCR-based molecular identification technique to examine bacterial assemblage composition in background aerosols of the eastern Mediterranean Sea during a rapid change of meteorological conditions. 16S rDNA fragments of 166 clones were identified and were affiliated with six bacterial phyla (Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, Spirochetes, and Fusobacteria), plant-related sequences, and other uncultivated bacterial groups. The analyzed clones were closely related to sequences previously characterized from diverse sources including soil, plants, marine water and sediment, human skin, activated sludge, house dust, indoor air, gut microbiota, and food. Plant- and human-associated sequences accounted for the largest fraction of the identified clones. Spore-forming Firmicutes showed a considerable increase when air mass origin changed from north to south implying that south winds favored bacterial spores transportation from the inland of Crete or North Africa. However, no conclusive trends were revealed for other groups of microorganisms. The influence of air mass origin was further investigated for marine- and terrestrial-associated sequences. A higher number of marine-associated sequences were identified when south winds crossed the inland of Crete, while the opposite was observed when north winds passed over the Aegean Sea. This discrepancy could be partly explained by the fact that the north winds were blowing at very low speed which constrained the formation of sea-spray aerosols and the ejection of marine microbes from sea surface to the atmosphere. Overall, the interpretation of bacterial assemblage composition in relation to the meteorological conditions was proved to be a complicated task which is in line with previous studies.     

Determining the mutagenic activity of a tar, its vapors and aerosols

The Ames test was performed on Salmonella typhimurium, strain TA98, TA100, TA1535, TA1537, TA1538, to evaluate the mutagenic potential of a tar as well as its vapors and aerosols emitted at 250, 350 and 550 degrees C. Two chemical procedures were used: extractions of aromatics for DMSO; elimination of acids, alcohols and phenols. Weak mutagenic activity was demonstrated at each temperature. Then, using only Salmonella typhimurium strains TA98 and TA100, a study was made on the effects of the mutagenic compounds, benzo[a]pyrene, 2-aminoanthracene, nitrofluorene, methyl methanesulfonate and on the vapors and aerosols emitted at 350 degrees C by road-coating tar. For promutagenic compounds, an enhancing effect was observed before an inhibition effect. For direct mutagenic compounds, only the inhibition effect appeared. The mutagenic and/or carcinogenic activity was usually tested on a pure isolated chemical compound.     

Seasonal Changes in Endotoxin Exposure and Its Relationship to Exhaled Nitric Oxide and Exhaled Breath Condensate pH Levels in Atopic and Healthy Children

Endotoxin, a component of the cell walls of gram-negative bacteria, is a contaminant in organic dusts (house dust) and aerosols. In humans, small amounts of endotoxin may cause a local inflammatory response. Exhaled nitric oxide (eNO) levels, an inflammation indicator, are associated with the pH values of exhaled breath condensate (EBC). This study evaluated seasonal changes on indoor endotoxin concentrations in homes and the relationships between endotoxin exposure and eNO/EBC pH levels for healthy children and children with allergy-related respiratory diseases. In total, 34 children with allergy-related respiratory diseases and 24 healthy children were enrolled. Indoor air quality measurements and dust sample analysis for endotoxin were conducted once each season inside 58 surveyed homes. The eNO, EBC pH levels, and pulmonary function of the children were also determined. The highest endotoxin concentrations were on kitchen floors of homes of children with allergy-related respiratory diseases and healthy children, and on bedroom floors of homes of asthmatic children and healthy children. Seasonal changes existed in endotoxin concentrations in dust samples from homes of children with allergic rhinitis, with or without asthma, and in EBC pH values among healthy children and those with allergy-related respiratory diseases. Strong relationships existed between endotoxin exposure and EBC pH values in children with allergic rhinitis.