Cell Equivalents Research Articles

Development of an on-disc isothermal in vitro amplification and detection of bacterial RNA

We present a centrifugal microfluidic “Lab-on-a-Disc” (LoaD) system capable of implementing nucleic acid in vitro amplification using non-contact heating and fluorescence detection. The system functionality is verified by implementing a Nucleic Acid Sequence Based Amplification (NASBA) reaction, targeting the tmRNA transcript of Haemophilus influenzae. The NASBA assay incorporates fluorescent molecular beacon probes reporting target tmRNA amplification for endpoint detection. The system implements non-contact IR heating to heat the NASBA reaction to the required target temperatures during denaturation and amplification steps. The LoaD control system facilitates spin speed and chamber positioning for heating and fluorescence detection. The LoaD alignment system uses magnetic fields to locate and lock the chamber in the required position (heating or detection). The NASBA assay was implemented on the system using Haemophilus influenzae tmRNA over the range 102–104 cell equivalent (CE) units. For comparison, identical qNASBA assays were implemented on a Roche LightCycler 2.0 over this concentration range.

Historical Associations of Molecular Measurements of Escherichia coli and Enterococci to Anthropogenic Activities and Climate Variables in Freshwater Sediment Cores.

This study investigated the long-term associations of anthropogenic (sedimentary P, C, and N concentrations, and human population in the watershed), and climatic variables (air temperature, and river discharge) with Escherichia coli uidA and enterococci 23S rRNA concentrations in sediment cores from Anchor Bay (AB) in Lake St. Clair, and near the mouth of the Clinton River (CR), Michigan. Calendar year was estimated from vertical abundances of (137)Cs. The AB and CR cores spanned c.1760-2012 and c.1895-2012, respectively. There were steady state concentrations of enterococci in AB during c.1760-c.1860 and c.1910-c.2003 at ∼0.1 × 10(5) and ∼2.0 × 10(5) cell equivalents (CE) per g-dry wt, respectively. Enterococci concentrations in CR increased toward present day, and ranged from ∼0.03 × 10(5) to 9.9 × 10(5) CE/g-dry wt. The E. coli concentrations in CR and AB increased toward present day, and ranged from 0.14 × 10(7) to 1.7 × 10(7) CE/g-dry wt, and 1.8 × 10(6) to 8.5 × 10(6) CE/g-dry wt, respectively. Enterococci was associated with population and river discharge, while E. coli was associated with population, air temperature, and N and C concentrations (p < 0.05). Sediments retain records of the abundance of fecal indicator bacteria, and offer a way to evaluate responses to increased population, nutrient loading, and environmental policies.

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans.

Fungal DNA in dust in Swedish day care centres: associations with respiratory symptoms, fractional exhaled nitrogen oxide (FeNO) and C-reactive protein (CRP) in serum among day care centre staff.

To study associations between fungal DNA in day care centres, fractional exhaled nitric oxide (FeNO) and inflammatory markers in day care centre staff. Totally, 62 staff (90 %) from five day care centres in Sweden participated. All were females. Settled dust was collected and analysed for five sequences of fungal DNA by quantitative PCR. Levels of FeNO (NIOX MINO 50 ml/min) and serum levels of eosinophilic cationic protein, myeloperoxidase (MPO) and high-sensitivity C-reactive protein in blood (HsCRP) were measured. Dynamic spirometry was performed, and dyspnoea was measured. Biomarkers and dyspnoea ratings were log-transformed, and associations were analysed by linear mixed models, adjusting for age, atopy, smoking, body mass index (BMI), ETS and dampness/mould at home. Geometric mean (GM) for FeNO was 15.3 ppb, 6% were smokers, 14% were obese, 31% were overweight and 18% had atopy. GM concentration was 2.16 × 10(5) cell equivalents (CE)/g for total fungal DNA, 2310 CE/g for Aspergillus/penicillium (Asp/Pen) DNA, 17 CE/g for Aspergillus versicolor DNA and 14 CE/g dust for Streptomyces DNA. FeNO was associated with total fungal DNA (p = 0.004), Asp/Pen DNA (p = 0.005) and Streptomyces DNA (p = 0.03). HsCRP was associated with total fungal DNA (p = 0.03) and BMI (p = 0.001). Dyspnoea was associated with Asp/Pen DNA (p = 0.04). Subjects with ETS at home had lower lung function (FEV1) (p = 0.03), and those with dampness/mould at home had lower MPO (p = 0.03). Fungal contamination in day care centres, measured as fungal DNA, can be a risk factor for airway inflammation, and CRP is associated with BMI.

Molecular survey of occurrence and quantity of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa and amoeba hosts in municipal drinking water storage tank sediments

To examine the occurrence and quantity of potential pathogens and an indicator of microbial contamination in the sediments of municipal drinking water storage tanks (MDWSTs), given the absence of such data across the United States. Sediment samples (87 MDWST) from eighteen locations across ten states of the United States were collected and assayed by qPCR for a range of potential enteric and opportunistic microbial pathogens and a sewage-associated Bacteroides marker. Potential opportunistic pathogens dominated, with the highest detection of occurrence (per cent positive detection; average cell equivalence (CE)) being Mycobacterium spp. (88·9%; 6·7 ± 8·5 × 10(4) CE g(-1) ), followed by Legionella spp. (66·7%; 5·2 ± 5·9 × 10(3) CE g(-1) ), Pseudomonas aeruginosa (22·2%; 250 ± 880 CE g(-1) ) and Acanthamoeba spp. (38·9%; 53 ± 70 CE g(-1) ), with no detected Naegleria fowleri. Most enteric pathogens (Campylobacter jejuni, Escherichia coli 0157:H7, Salmonella enterica, Cryptosporidium parvum and Giardia duodenalis) were not detected, except for a trace signal for Campylobacter spp. There was significant correlation between the qPCR signals of Legionella spp. and Acanthamoeba spp. (R(2) = 0·61, n = 87, P = 0·0001). Diverse Legionella spp. including Leg. pneumophila, Leg. pneumophila sg1 and Leg. anisa were identified, each of which might cause legionellosis. These results imply that potential opportunistic pathogens are common within MDWST sediments and could act as a source of microbial contamination, but need downstream growth to be of potential concern. The results imply that opportunistic pathogen risks may need to be managed by regular tank cleaning or other management practices.

Use of Quantitative Real-Time PCR for Direct Detection of Serratia marcescens in Marine and Other Aquatic Environments

Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml(-1) and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml(-1). This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.

Comparisons of statistical models to predict fecal indicator bacteria concentrations enumerated by qPCR- and culture-based methods

Recently, the United States Environmental Protection Agency (USEPA) revised their recreational water quality criteria, in which adjustments were made by approving enterococci (ENT) quantitative PCR (qPCR) as an alternative, rapid method and advocating the use of predictive models for water quality management. The implementation of qPCR-based methods and prediction models are meant to decrease the time between sample collection and public advisories and notifications. To date, few studies have compared qPCR-based models to culture-based prediction models and none of these studies have been conducted in coastal estuarine systems. In this study, we created prediction models using qPCR-based fecal indicator bacteria (FIB) data in dual-use recreational and shellfish harvesting waters and compared them to published ENT and Escherichia coli (EC) culture-based prediction models in eastern North Carolina estuaries. Furthermore, an empirical statistical model was created to predict qPCR inhibition levels so that proper remediation techniques can be applied when it is a problem. Predictor variable selection in both qPCR- and culture-based ENT models was very similar; both models included 14-day rain total, dissolved oxygen, and salinity/conductivity, with 89 and 90% of qPCR and culture data described, respectively. Using ENT management action thresholds, qPCR- and culture-based models showed high accuracy in management decisions. The qPCR model had 92 and 96% accuracy using the 110 and 1000 cell equivalents (CE)/100 ml thresholds, respectively, and the culture model had 90% accuracy in management decisions with the 110 MPN/100 ml threshold. EC models for qPCR- and culture-based concentrations used similar independent variables (14-day humidity, salinity/conductivity, a rain/storm variable, and a measure of air temperature), with each model explaining 26 and 55% of the data variation, respectively. When using different thresholds that were logs apart for management decisions, the two EC models accurately predicted management decisions; qPCR models correctly predicted management decisions 96 and 77% of the time (using 31 and 320 CE/100 ml, respectively) while culture models correctly predicted management decisions 96 and 88% percent of the time (with 31 and 320 MPN/100 ml, respectively). Equivalency between models was shown in our non-point source impacted estuaries, with ENT models performing slightly better than EC models. In addition, inhibition of the qPCR was a major issue that had to be addressed. An inhibition model was created with easily obtained meteorological data and accounted for a high level of data variability (adjusted R2 = 0.82).

Mycobacterium aviumsubspeciesparatuberculosisis widely distributed inBritish soils and waters: implications for animal and human health

In the first comprehensive geographical survey of distribution in Great Britain, Mycobacterium avium ssp. paratuberculosis (MAP) was detected in 115 of 1092 (10.5%) soil cores, in the range of 5 × 10(2) to 3 × 10(6) MAP cell equivalents (CE) g(-1) wet weight soil with the majority of the positive PCR reactions (n = 75; 65%) occurring around the limit of detection (500-5000 CE g(-1) wet weight soil). The distribution of MAP significantly increased from North to South and was significantly correlated with increasing cattle numbers over the same longitudinal axis. Similarly MAP occurrence significantly increased towards easterly latitudes although none of the parameters measured were associated. Comparisons of land use indicated that MAP was widely distributed in both farming and non-farming areas. Soil core samples taken from the rivers Wyre and Douglas catchments (Lancashire, UK) and river Tywi (South Wales) were negative for MAP. However, river monitoring showed a consistent presence of MAPs throughout those catchments over a 6-month period. We concluded that MAP is widely distributed within and outside the confines of the farming environment; its geographical distribution is wider than originally anticipated and; monitoring rivers describes the MAP status of catchment better than individual soil samples.

Quantitative assessment of Naegleria fowleri and Escherichia coli concentrations within a Texas reservoir

Previous presence/absence studies have indicated a correlation between the presence of the pathogenic amoeba Naegleria fowleri and the presence of bacteria, such as the fecal indicator Escherichia coli, in environmental surface waters. The objective of this study was to use quantitative real-time polymerase chain reaction (qPCR) methodologies to measure N. fowleri and E. coli concentrations within a Texas reservoir in late summer, and to determine if concentrations of N. fowleri and E. coli were statistically correlated. N. fowleri was detected in water samples from 67% of the reservoir sites tested, with concentrations ranging up to an estimated 26 CE (cell equivalents)/100 mL. E. coli was detected in water samples from 60% of the reservoir sites tested, with concentrations ranging up to 427 CE/100 mL. In this study, E. coli concentrations were not indicative of N. fowleri concentrations.

A Real‐Time Polymerase Chain Reaction Assay for Quantification of Edwardsiella ictaluri in Catfish Pond Water and Genetic Homogeneity of Diagnostic Case Isolates from Mississippi

A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri-specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.

Implementation of a Quantitative Real-Time PCR Assay for the Detection of Mycobacterium immunogenum in Metalworking Fluids

The bacterium Mycobacterium immunogenum has been implicated in causing the lung condition hypersensitivity pneumonitis (HP) in factory workers exposed to colonized metalworking fluids (MWFs). M. immunogenum-specific, real-time quantitative PCR detection technique (MiRT-qPCR) was implemented on a large scale to 363 MWFs of varying types, originating from the United States and Europe, that had been in use for between 30 days and 1 year. In MWFs that contained between 103 and 109 culturable general heterotrophs mL−1 the technique detected between 5 and 2 × 106 mL−1 M. immunogenum cell equivalents (CE) in 12.2% (23 of 189) of U.S. samples and between 8 and 6 × 105 mL−1 CE in 39.1% (68 of 174) of samples from Europe. In contrast, only three cultured presumptive mycobacterial isolates across all samples were confirmed as M. immunogenum. Implementation of the assay demonstrated its practicality and further emphasized the limitations of using cultivation alone. Interestingly, no M. immunogenum were detected in mineral oil-based Bio-Concept MWFs from the United States, while it was more commonly detected in used MWFs based on formaldehyde-releasing biocides than in MWFs free of formaldehyde-depot biocides.

Sweeping Plaques From Brain Studied as Alzheimer's Strategy

Back to table of contents Previous article Next article Clinical & Research NewsFull AccessSweeping Plaques From Brain Studied as Alzheimer's StrategyJoan Arehart-TreichelJoan Arehart-TreichelSearch for more papers by this authorPublished Online:6 May 2011https://doi.org/10.1176/pn.46.9.psychnews_46_9_18_1AbstractEnormous efforts have been expended in the search to discover an effective treatment for Alzheimer's disease that involves inhibiting the production of plaques in the brain- that is, by targeting the enzymes that make amyloid protein. But all of the candidate drugs that once showed promise have proven disappointing in clinical trials. Therefore another tack for treating the disease may be needed- for example, the removal of plaques from the brain after they have already formed. Indeed, scientists at Rockefeller University have found a molecule that can do just that- at least in cultured rodent neurons. The senior investigator was Paul Greengard, Ph.D., a professor and head of the Laboratory of Molecular and Cellular Neuroscience at Rockefeller University and a Nobel laureate in medicine. The lead investigator was Yuan Tian, Ph.D., a post-doctoral associate in Greengard's laboratory. Their findings were published online March 2 in the FASEB Journal, a publication of the Federation of American Societies for Experimental Biology. Cells contain a pathway for degrading long-lived, aggregated proteins in a process called autophagy. So Greengard and his colleagues wondered whether the artificial induction of autophagy might be used to remove from the brain the proteins of which Alzheimer's plaques are made. They tested various small-molecule enhancers of autophagy on cultured rodent neurons containing clumps of amyloid protein and found that one -SMER28- was successful in removing the clumps. "At this point it is not clear if SMER28 is appropriate for animal studies [because of issues surrounding] bioavailability and brain permeability, but we would like to test its impact using animal models," Greengard told Psychiatric News. Regarding human clinical trials with SMER28, "it is too early to predict" whether they will be possible, he said. A cell's autophagy process attacks a protein from which Alzheimer's plaques are made.Credit: Paul Greengard, Ph.D., et al.In any event, Greengard and his colleagues believe that their cell-culture success with SMER28 points to the potential of small-molecule enhancers of autophagy as effective treatments for Alzheimer's. Gerald Weissmann, M.D., editor-in-chief of the FASEB Journal, pointed out in an accompanying press release, "Autophagy has been called the cell's equivalent of urban renewal. The Rockefeller group shows that strategies to remove the blight in cells that causes Alzheimer's disease are not only worth pursuing, but so far appear to be very promising." Autophagy enhancers may hold treatment promise for some other neurodegenerative diseases as well, Greengard and his colleagues noted. For example, some small-molecule autophagy enhancers have been found to degrade, in cell culture, aggregates of mutant huntingtin proteins, the hallmark of Huntington's disease. And even more intriguingly, when mice were fed an autophagy enhancer called rapamycin, it extended their lifespan by about 10 percent, David Harrison, Ph.D., a professor at Jackson Laboratory in Bar Harbor, Maine, and colleagues reported in the July 16, 2009, Nature. "SMER28 stands for 'small-molecule-enhancer-of-rapamycin-28,' but it really has nothing to do with the actual rapamycin molecule," Greengard explained to Psychiatric News. "It is called that because it was initially discovered as an enhancer of the effect of rapamycin to stimulate autophagy. In fact, we now know that SMER28 works through a pathway independent of rapamycin." The research was funded by the National Institutes of Health and the Fisher Center for Alzheimer's Research Foundation.An abstract of "A Small-Molecule Enhancer of Autophagy Decreases Levels of Amyloid Beta and APP-CTF Via Atg5-Dependent Autophagy Pathway" is posted at <www.fasebj.org/content/early/2011/03/02/fj.10-175158.abstract>. ISSUES NewArchived

Fungal DNA, allergens, mycotoxins and associations with asthmatic symptoms among pupils in schools from Johor Bahru, Malaysia

While there is a large variation of prevalence of asthma symptoms worldwide, what we do know is that it is on the rise in developing countries. However, there are few studies on allergens, moulds and mycotoxin exposure in schools in tropical countries. The aims were to measure selected fungal DNA, furry pet allergens and mycotoxins in dust samples from schools in Malaysia and to study associations with pupils' respiratory health effects. Eight secondary schools and 32 classrooms in Johor Bahru, Malaysia were randomly selected. A questionnaire with standardized questions was used for health assessment in 15 randomly selected pupils from each class. The school buildings were inspected and both indoor and outdoor climate were measured. Dust samples were collected by cotton swabs and Petri dishes for fungal DNA, mycotoxins and allergens analysis. The participation rate was 96% (462/480 invited pupils), with a mean age of 14 yr (range 14-16). The pupils mostly reported daytime breathlessness (41%), parental asthma or allergy (22%), pollen or pet allergy (21%) and doctor-diagnosed asthma (13%) but rarely reported night-time breathlessness (7%), asthma in the last 12 months (3%), medication for asthma (4%) or smoking (5%). The inspection showed that no school had any mechanical ventilation system, but all classrooms had openable windows that were kept open during lectures. The mean building age was 16 yr (range 3-40) and the mean indoor and outdoor CO(2) levels were 492 ppm and 408 ppm, respectively. The mean values of indoor and outdoor temperature and relative humidity were the same, 29°C and 70% respectively. In cotton swab dust samples, the Geometric Mean (GM) value for total fungal DNA and Aspergillus/Penicillium (Asp/Pen) DNA in swab samples (Cell Equivalents (CE)/m(2)) was 5.7*10(8) and 0.5*10(8), respectively. The arithmetic mean (CE/m(2)) for Aspergillus versicolor DNA was 8780, Stachybotrys chartarum DNA was 26 and Streptomyces DNA was 893. The arithmetic means (pg/m(2)) for the mycotoxins sterigmatocystin and verrucarol were 2547 and 17, respectively. In Petri dish dust samples, the GM value for total fungal DNA and Asp/Pen DNA (CE/m(2) per day) was 9.2*10(6) and 1.6*10(6), respectively. The arithmetic mean (CE/m(2) per day) for A. versicolor DNA was 1478, S. chartarum DNA was 105 and Streptomyces DNA was 1271, respectively. The GM value for cat (Fel d1) allergen was 5.9 ng/m(2) per day. There were positive associations between A. versicolor DNA, wheeze and daytime breathlessness and between Streptomyces DNA and doctor-diagnosed asthma. However, the associations were inverse between S. chartarum DNA and daytime breathlessness and between verrucarol and daytime breathlessness. In conclusion, fungal DNA and cat allergen contamination were common in schools from Malaysia and there was a high prevalence of respiratory symptoms among pupils. Moreover, there were associations between levels of some fungal DNA and reported respiratory health in the pupils.

Linking non-culturable (qPCR) and culturable enterococci densities with hydrometeorological conditions

Quantitative polymerase chain reaction (qPCR) measurement of enterococci has been proposed as a rapid technique for assessment of beach water quality, but the response of qPCR results to environmental conditions has not been fully explored. Culture-based E. coli and enterococci have been used in empirical predictive models to characterize their responses to environmental conditions and to increase monitoring frequency and efficiency. This approach has been attempted with qPCR results only in few studies. During the summer of 2006, water samples were collected from two southern Lake Michigan beaches and the nearby river outfall (Burns Ditch) and were analyzed for enterococci by culture-based and non-culture-based (i.e., qPCR) methods, as well as culture-based E. coli. Culturable enterococci densities (log CFU/100 ml) for the beaches were significantly correlated with enterococci qPCR cell equivalents (CE) ( R = 0.650, P < 0.0001, N = 32). Enterococci CE and CFU densities were highest in Burns Ditch relative to the beach sites; however, only CFUs were significantly higher ( P < 0.0001). Culturable enterococci densities at Burns Ditch and the beaches were significantly correlated ( R = 0.565, P < 0.0001, N = 32). Culturable E. coli and enterococci densities were significantly correlated ( R = 0.682, P < 0.0001, N = 32). Regression analyses suggested that enterococci CFU could be predicted by lake turbidity, Burns Ditch discharge, and wind direction (adjusted R 2 = 0.608); enterococci CE was best predicted by Burns Ditch discharge and log-transformed lake turbidity × wave height (adjusted R 2 = 0.40). In summary, our results show that analytically, the qPCR method compares well to the non-culture-based method for measuring enterococci densities in beach water and that both these approaches can be predicted by hydrometeorological conditions. Selected predictors and model results highlight the differences between the environmental responses of the two method endpoints and the potentially high variance in qPCR results.

Abstract 2812: Chromosome translocation frequency and intakes of B vitamins, meat, and grain products in airline pilots

Abstract B vitamins such as folate and vitamins B2, B6, and B12 are essential for the maintenance of DNA integrity. Deficiency in these B vitamins can result in DNA strand breaks which, if unrepaired, can lead to the formation of chromosome aberrations (CA). Niacin also contributes to chromosome stability due to several important roles, including DNA repair and regulation of cell division and apoptosis. Animal and in vitro studies suggest that these B vitamins may protect against chromosome damage induced by ionizing radiation (IR), but data from IR-exposed human populations are limited. Airline pilots are exposed to cosmic IR, an efficient inducer of CA which have been shown to be associated with increased cancer risk. We examined the association between the frequency of translocations (stable form of CA) and intakes of these B vitamins and their food sources in 82 male airline pilots. All subjects completed a self-administered questionnaire with information on occupational history and lifestyle factors. Dietary vitamin B intakes were estimated using a validated semiquantitative food-frequency questionnaire and were energy-adjusted. Translocations were scored in an average of 1039 cell equivalents (CE) per subject using fluorescence in situ hybridization with whole chromosome paints, and expressed per 100 CE. Negative binomial regression was used to estimate rate ratios (RR) and 95% confidence intervals (CI), adjusted for age, flight experience in years, self-reported personal diagnostic X-ray procedures, military service, and lifestyle factors. We observed a significant inverse association between the frequency of translocations and intake of niacin from food: adjusted RR (95% CI) for subjects in the highest compared with lowest tertile was 0.58 (0.40, 0.83). Translocation frequency was not associated with total niacin intake from food and supplements, nor was it associated with intakes of folate, or vitamin B2, B6, or B12 either from food or from food and supplements. These results remained unchanged after further adjustment for the combined intakes of vitamins C and E, β-carotene, β-cryptoxanthin, and lutein-zeaxanthin from food (previously shown to be significantly and inversely associated with translocation frequency), or multivitamin supplement use. Additionally, the adjusted RR (95% CI) for ≥ median compared with &amp;lt; median intake of whole grains (median 7.5 servings/week) was 0.69 (0.50, 0.96) and of red and processed meat (median 14.5 servings/week) was 1.56 (1.13, 2.16). Significant inverse associations were also previously observed for high intakes of vitamin C-rich fruit and vegetables, citrus fruit, and green leafy vegetables which are food sources of B vitamins, especially folate. Our data suggest that a high intake of niacin from food or a diet high in whole grains as well as fruits and vegetables but low in red and processed meat may protect against IR-induced chromosome damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2812.

Increased frequency of chromosome translocations in airline pilots with long-term flying experience

Background: Chromosome translocations are an established biomarker of cumulative exposure to external ionising radiation. Airline pilots are exposed to cosmic ionising radiation, but few flight crew studies have examined translocations...

High Sensitivity of Children to Swimming-Associated Gastrointestinal Illness

Culture-based methods of monitoring fecal pollution in recreational waters require 24 to 48 hours to obtain results. This delay leads to potentially inaccurate management decisions regarding beach safety. We evaluated the quantitative polymerase chain reaction (QPCR) as a faster method to assess recreational water quality and predict swimming-associated illnesses. We enrolled visitors at 4 freshwater Great Lakes beaches, and contacted them 10 to 12 days later to ask about health symptoms experienced since the visit. Water at the beaches was polluted by point sources that carried treated sewage. We tested water samples daily for Enterococcus using QPCR and membrane filtration (EPA Method 1600). We completed 21,015 interviews and tested 1359 water samples. Enterococcus QPCR cell equivalents (CEs) were positively associated with swimming-associated gastrointestinal (GI) illness (adjusted odds ratio per 1 log10 QPCR CE =1.26; 95% confidence interval = 1.06-1.51). The association between GI illness and QPCR CE was stronger among children aged 10 years and below (1.69; 1.24-2.30). Nonenteric illnesses were not consistently associated with Enterococcus QPCR CE exposure, although rash and earache occurred more frequently among swimmers. Enterococcus QPCR CE exposure was more strongly associated with GI illness than Enterococcus measured by membrane filtration. Measurement of the indicator bacteria Enterococci in recreational water using a rapid QPCR method predicted swimming-associated GI illness at freshwater beaches polluted by sewage discharge. Children at 10 years or younger were at greater risk for GI illness following exposure.

Detection of Mycobacterium immunogenum by real-time quantitative Taqman PCR

A quantitative real-time 5′-nuclease (Taqman) PCR technique was developed to specifically detect Mycobacterium immunogenum. rpoB-specific primers and Taqman probe were evaluated for detection of M. immunogenum DNA extracted from pure cultures and from industrial metal working fluids (MWFs). Specificity was confirmed and the sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). When tested on industrial metal working fluids from the UK and USA from which no M. immunogenum CFU were recovered, the assay detected between 3.4 × 10 1 and 1.9 × 10 4 cell equivalents (CE) per ml, and increased the detection rate over culture to 37.5% (12 of 32 samples). This assay provides a specific, sensitive and rapid method for the detection of M. immunogenum and is applicable within industry for the early detection of this human pathogen and to the possible prevention of hypersensitivity pneumonitis (HP) in workers.

Retrospective Biodosimetry among United States Radiologic Technologists

Measurement of chromosome translocations in peripheral blood lymphocytes has been used to quantify prior exposure to ionizing radiation, including for workers exposed to low, chronic doses. We assessed translocation frequencies in a subset of U.S. radiologic technologists to substantiate ionizing radiation dose estimates developed for 110,418 technologists who worked between 1916 and 1984. From 3,441 cohort members known to have begun working before 1950, we selected a sample of 152, stratified by estimated cumulative dose, over-sampling from higher-dose categories and excluding persons with a prior cancer diagnosis, a personal or family history of chromosomal instability disorders, or a current history of smoking. Estimates of film-badge dose ranged from less than 10 cSv to more than 30 cSv. Blood samples, obtained in 2004, were analyzed by fluorescence in situ hybridization (FISH) whole chromosome painting by simultaneously labeling chromosomes 1, 2 and 4 in red and 3, 5 and 6 in green. Translocations were scored in 1800 well-spread metaphase cells and expressed per 100 cell equivalents (CE) per person. Linear Poisson regression models with allowance for overdispersion were used to assess the relationship between estimated occupational red bone marrow absorbed dose in cGy and translocation frequency, adjusted for age, gender and estimated red bone marrow absorbed dose score from personal diagnostic procedures. We observed 0.09 excess translocations per 100 CE per cGy red bone marrow dose (95% CI: -0.01, 0.2; P = 0.07), which is similar to the expected estimate based on previous cytogenetic studies (0.05 excess translocations per 100 CE per cGy). Despite uncertainty in the estimates of occupational red bone marrow absorbed doses, we found good general agreement between the doses and translocation frequencies, lending support to the credibility of the dose assessment for this large cohort of U.S. radiologic technologists.

Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets.

To determine whether Megasphaera elsdenii YE34 (lactic acid degrader) and Butyrivibrio fibrisolvens YE44 (alternative starch utilizer to Streptococcus bovis) establish viable populations in the rumen of beef cattle rapidly changed from a forage-based to a grain-based diet. Five steers were inoculated with the two bacterial strains (YE34 and YE44) and five served as uninoculated controls. With the exception of one animal in the control group, which developed acidosis, all steers rapidly adapted to the grain-based diet without signs of acidosis (pH decline and accumulation of lactic acid). Bacterial populations of S. bovis, B. fibrisolvens and M. elsdenii were enumerated using real-time Taq nuclease assays. Populations of S. bovis remained constant (except in the acidotic animal) at ca 10(7) cell equivalents (CE) ml-1 throughout the study. Megasphaera elsdenii YE34, was not detectable in animals without grain in the diet, but immediately established in inoculated animals, at 10(6) CE ml-1, and increased 100-fold in the first 4 days following inoculation. Butyrivibrio fibrisolvens, initially present at 10(8) CE ml-1, declined rapidly with the introduction of grain into the diet and was not detectable 8 days after grain introduction. Megasphaera elsdenii rapidly establishes a lactic acid-utilizing bacterial population in the rumen of grain-fed cattle 7-10 days earlier than in uninoculated cattle. The study has demonstrated that rumen bacterial populations, and in particular the establishment of bacteria inoculated into the rumen for probiotic use, can be monitored by real-time PCR.