Evidence of Avian and Possum Fecal Contamination in Rainwater Tanks as Determined by Microbial Source Tracking Approaches.

UNLABELLED: Avian and possum fecal droppings may negatively impact roof-harvested rainwater (RHRW) water quality due to the presence of zoonotic pathogens. This study was aimed at evaluating the performance characteristics of a possum feces-associated (PSM) marker by screening 210 fecal and wastewater samples from possums (n = 20) and a range of nonpossum hosts (n = 190) in Southeast Queensland, Australia. The host sensitivity and specificity of the PSM marker were 0.90 and 0.95 (maximum value, 1.00), respectively. The mean concentrations of the GFD marker in possum fecal DNA samples (8.8 × 10(7) gene copies per g of feces) were two orders of magnitude higher than those in the nonpossum fecal DNA samples (5.0 × 10(5) gene copies per g of feces). The host sensitivity, specificity, and concentrations of the avian feces-associated GFD marker were reported in our recent study (W. Ahmed, V. J. Harwood, K. Nguyen, S. Young, K. Hamilton, and S. Toze, Water Res 88:613-622, 2016, http://dx.doi.org/10.1016/j.watres.2015.10.050). The utility of the GFD and PSM markers was evaluated by testing a large number of tank water samples (n = 134) from the Brisbane and Currumbin areas. GFD and PSM markers were detected in 39 of 134 (29%) and 11 of 134 (8%) tank water samples, respectively. The GFD marker concentrations in PCR-positive samples ranged from 3.7 × 10(2) to 8.5 × 10(5) gene copies per liter, whereas the concentrations of the PSM marker ranged from 2.0 × 10(3) to 6.8 × 10(3) gene copies per liter of water. The results of this study suggest the presence of fecal contamination in tank water samples from avian and possum hosts. This study has established an association between the degradation of microbial tank water quality and avian and possum feces. Based on the results, we recommend disinfection of tank water, especially for tanks designated for potable use. IMPORTANCE: The use of roof-harvested rainwater (RHRW) for domestic purposes is a globally accepted practice. The presence of pathogens in rainwater tanks has been reported by several studies, supporting the necessity for the management of potential health risks. The sources of fecal pollution in rainwater tanks are unknown. However, the application of microbial source tracking (MST) markers has the potential to identify the sources of fecal contamination in a rainwater tank. In this study, we provide evidence of avian and possum fecal contamination in tank water samples using molecular markers. This study established a potential link between the degradation of the microbial quality of tank water and avian and possum feces.

Public health implications of Acanthamoeba and multiple potential opportunistic pathogens in roof-harvested rainwater tanks.

A study of six potential opportunistic pathogens (Acanthamoeba spp., Legionella spp., Legionella longbeachae, Pseudomonas aeruginosa, Mycobacterium avium and Mycobacterium intracellulare) and an accidental human pathogen (Legionella pneumophila) in 134 roof-harvested rainwater (RHRW) tank samples was conducted using quantitative PCR (qPCR). All five opportunistic pathogens and accidental pathogen L. pneumophila were detected in rainwater tanks except Legionella longbeachae. Concentrations ranged up to 3.1×10(6) gene copies per L rainwater for Legionella spp., 9.6×10(5) gene copies per L for P. aeruginosa, 6.8×10(5) gene copies per L for M. intracellulare, 6.6×10(5) gene copies per L for Acanthamoeba spp., 1.1×10(5) gene copies per L for M. avium, and 9.8×10(3) gene copies per L for L. pneumophila. Among the organisms tested, Legionella spp. (99% tanks) were the most prevalent followed by M. intracellulare (78%). A survey of tank-owners provided data on rainwater end-uses. Fecal indicator bacteria (FIB) Escherichia coli and Enterococcus spp. were enumerated using culture-based methods, and assessed for correlations with opportunistic pathogens and L. pneumophila tested in this study. Opportunistic pathogens did not correlate well with FIB except E. coli vs. Legionella spp. (tau=0.151, P=0.009) and E. coli vs. M. intracellulare (tau=0.14, P=0.015). However, M. avium weakly correlated with both L. pneumophila (Kendall's tau=0.017, P=0.006) and M. intracellulare (tau=0.088, P=0.027), and Legionella spp. also weakly correlated with M. intracellulare (tau=0.128, P=0.028). The presence of these potential opportunistic pathogens in tank water may present health risks from both the potable and non-potable uses documented from the current survey data.

Wastewater-based epidemiology as a public health resource in low- and middle-income settings.

In the face of emerging and re-emerging diseases, novel and innovative approaches to population scale surveillance are necessary for the early detection and quantification of pathogens. The last decade has seen the rapid development of wastewater and environmental surveillance (WES) to address public health challenges, which has led to establishment of wastewater-based epidemiology (WBE) approaches being deployed to monitor a range of health hazards. WBE exploits the fact that excretions and secretions from urine, and from the gut are discharged in wastewater, particularly sewage, such that sampling sewage systems provides an early warning system for disease outbreaks by providing an early indication of pathogen circulation. While WBE has been mainly used in locations with networked wastewater systems, here we consider its value for less connected populations typical of lower-income settings, and in assess the opportunity afforded by pit latrines to sample communities and localities. We propose that where populations struggle to access health and diagnostic facilities, and despite several additional challenges, sampling unconnected wastewater systems remains an important means to monitor the health of large populations in a relatively cost-effective manner.

High resolution data visualization and machine learning prediction of free chlorine residual in a green building water system.

People spend most of their time indoors and are exposed to numerous contaminants in the built environment. Water management plans implemented in buildings are designed to manage the risks of preventable diseases caused by drinking water contaminants such as opportunistic pathogens (e.g., Legionella spp.), metals, and disinfection by-products (DBPs). However, specialized training required to implement water management plans and heterogeneity in building characteristics limit their widespread adoption. Implementation of machine learning and artificial intelligence (ML/AI) models in building water settings presents an opportunity for faster, more widespread use of data-driven water quality management approaches. We demonstrate the utility of Random Forest and Long Short-Term Memory (LSTM) ML models for predicting a key public health parameter, free chlorine residual, as a function of data collected from building water quality sensors (ORP, pH, conductivity, and temperature) as well as WiFi signals as a proxy for building occupancy and water usage in a "green" Leadership in Energy and Environmental Design (LEED) commercial and institutional building. The models successfully predicted free chlorine residual declines below 0.2 ppm, a common minimum reference level for public health protection in drinking water distribution systems. The predictions were valid up to 5 min in advance, and in some cases reasonably accurate up to 24 h in advance, presenting opportunities for proactive water quality management as part of a sense-analyze-decide framework. An online data dashboard for visualizing water quality in the building is presented, with the potential to link these approaches for real-time water quality management.

EMA-amplicon-based sequencing informs risk assessment analysis of water treatment systems.

Illumina amplicon-based sequencing was coupled with ethidium monoazide bromide (EMA) pre-treatment to monitor the total viable bacterial community and subsequently identify and prioritise the target organisms for the health risk assessment of the untreated rainwater and rainwater treated using large-volume batch solar reactor prototypes installed in an informal settlement and rural farming community. Taxonomic assignments indicated that Legionella and Pseudomonas were the most frequently detected genera containing opportunistic bacterial pathogens in the untreated and treated rainwater at both sites. Additionally, Mycobacterium, Clostridium sensu stricto and Escherichia/Shigella displayed high (≥80%) detection frequencies in the untreated and/or treated rainwater samples at one or both sites. Numerous exposure scenarios (e.g. drinking, cleaning) were subsequently investigated and the health risk of using untreated and solar reactor treated rainwater in developing countries was quantified based on the presence of L. pneumophila, P. aeruginosa and E. coli. The solar reactor prototypes were able to reduce the health risk associated with E. coli and P. aeruginosa to below the 1 × 10-4 annual benchmark limit for all the non-potable uses of rainwater within the target communities (exception of showering for E. coli). However, the risk associated with intentional drinking of untreated or treated rainwater exceeded the benchmark limit (E. coli and P. aeruginosa). Additionally, while the solar reactor treatment reduced the risk associated with garden hosing and showering based on the presence of L. pneumophila, the risk estimates for both activities still exceeded the annual benchmark limit. The large-volume batch solar reactor prototypes were thus able to reduce the risk posed by the target bacteria for non-potable activities rainwater is commonly used for in water scarce regions of sub-Saharan Africa. This study highlights the need to assess water treatment systems in field trials using QMRA.

Outbreaks of Legionnaires' Disease and Pontiac Fever 2006-2017.

PURPOSE OF REVIEW: The global importance of Legionnaires' disease (LD) and Pontiac fever (PF) has grown in recent years. While sporadic cases of LD and PF do not always provide contextual information for evaluating causes and drivers of Legionella risks, analysis of outbreaks provides an opportunity to assess these factors. RECENT FINDINGS: A review was performed and provides a summary of LD and PF outbreaks between 2006 and 2017. Of the 136 outbreaks, 115 were LD outbreaks, 4 were PF outbreaks, and 17 were mixed outbreaks of LD and PF. Cooling towers were implicated or suspected in the a large portion of LD or PF outbreaks (30% total outbreaks, 50% confirmed outbreak-associated cases, and 60% outbreak-associated deaths) over this period of time, while building water systems and pools/spas were also important contributors. Potable water/building water system outbreaks seldom identify specific building water system or fixture deficiencies. The outbreak data summarized here provides information for prioritizing and targeting risk analysis and mitigation strategies.

Amplicon-based taxonomic characterization of bacteria in urban and peri-urban roof-harvested rainwater stored in tanks.

Overall, 26% of Australian households use rainwater tanks as a source of potable and nonpotable water. Limited information is available on the total bacterial communities in tank water. Therefore, identification of dominant bacterial communities, diversity, and their distribution is important in understanding the microbial quality of tank water. In this study, the abundance and diversity of bacterial communities in 88 tank water samples collected from the urban areas of Brisbane (n=44) and the peri-urban center of Currumbin (n=44) in Southeast Queensland, Australia were determined using amplicon-based Illumina next-generation sequencing. In addition, the SourceTracker program was used to identify the sources of fecal contamination in tank water samples. Sequence reads were also analyzed to detect potential bacterial pathogenic genera in the tank water samples collected. Differences in sample coverage, alpha diversity, and richness did not differ significantly between the Brisbane and Currumbin tank water samples. Comamonadaceae and Planctomycetaceae were the most abundant families in all tank water samples. Curvibacter was the most abundant genus in all tank water samples. SourceTracker revealed that around 34% (Brisbane) and 43% (Currumbin) of tank water samples had a signature for bird fecal contamination. The potential opportunistic pathogenic genera including Burkholderia, Chromobacterium, Clostridium, Legionella, Mycobacterium, Nocardia, and Pseudomonas were most prevalent in tank water samples. Next-generation sequencing can be used as an initial screening tool to identify a wide array of potential pathogenic genera in tank water samples followed by quantifying specific pathogen(s) of interest using more sensitive molecular assays such as quantitative PCR (qPCR).

Properties of external plexiform layer interneurons in mouse olfactory bulb slices.

In the external plexiform layer (EPL) of the main olfactory bulb, apical dendrites of inhibitory granule cells form large numbers of synapses with mitral and tufted (M/T) cells, which regulate the spread of activity along the M/T cell dendrites. The EPL also contains intrinsic interneurons, the functions of which are unknown. In the present study, recordings were obtained from cell bodies in the EPL of mouse olfactory bulb slices. Biocytin-filling confirmed that the recorded cells included interneurons, tufted cells, and astrocytes. The interneurons had fine, varicose dendrites, and those located superficially bridged the EPL space below several adjacent glomeruli. Interneuron activity was characterized by high frequency spontaneous excitatory postsynaptic potential/currents that were blocked by the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and largely eliminated by the voltage-sensitive Na+ channel blocker, tetrodotoxin. Interneuron activity differed markedly from that of tufted cells, which usually exhibited spontaneous action potential bursts. The interneurons produced few action potentials spontaneously, but often produced them in response to depolarization and/or olfactory nerve (ON) stimulation. The responses to depolarization resembled responses of late- and fast-spiking interneurons found in other cortical regions. The latency and variability of the ON-evoked responses were indicative of polysynaptic input. Interneurons expressing green fluorescent protein under control of the mouse glutamic acid decarboxylase 65 promoter exhibited identical properties, providing evidence that the EPL interneurons are GABAergic. Together, these results suggest that EPL interneurons are excited by M/T cells via AMPA/kainate receptors and may in turn inhibit M/T cells within spatial domains that are topographically related to several adjacent glomeruli.

Distribution of immunoreactivity for gamma-aminobutyric acid in the salamander olfactory bulb.

Intrinsic neurons provide inhibitory synaptic input to mitral (and tufted) output cells within several laminae of the olfactory bulb. In rodents, the two main types of intrinsic neurons are granule and periglomerular cells, both of which contain gamma-aminobutyric acid (GABA). In the present study, immunocytochemical techniques were used to determine whether intrinsic neurons in the salamander olfactory bulb might also contain GABA. With the aid of two antisera to different GABA-conjugates, immunoreactivity for GABA was localized within the olfactory bulb laminae. In the glomerular layer, periglomerular cells, which were strongly immunoreactive, were concentrated in clusters along the border with the olfactory nerve layer. Dendrites of the cells encircled nearby glomeruli and were presumably a primary source of intraglomerular processes that were also stained. In the subglomerular region and external plexiform layer, relatively few immunoreactive cells were observed, most of which appeared to be periglomerular and tufted cell types with glomerular dendrites. Throughout the external plexiform and mitral cell layers, however, a dense matrix of spiny processes and puncta was stained, outlining large, unstained dendrites derived from the large, unstained cell bodies of mitral cells. The spiny processes and puncta appeared to be derived from granule cells, which were the most abundant immunoreactive cells in the bulb. Granule cell bodies filled the granule cell layer. In tissue fixed with 0.1-0.2% glutaraldehyde, staining in the olfactory bulb laminae was blocked by preadsorption of the two antisera with glutaraldehyde-conjugated GABA-bovine serum albumin. The staining therefore appeared to be specific for fixed GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Design and in vivo analysis of potent non-thiol inhibitors of farnesyl protein transferase.

Inhibitors of farnesyl protein transferase (FPTase) based upon a pseudotripeptide template are described that comprise an imidazole group substituted with a hydrophobic substituent. (1, 5)-Disubstitution of the imidazole group is shown to be the optimal array that leads to potent and selective inhibitors of FPTase. A variety of aryl and isoprenyl substituents are shown to afford effective inhibitors, and the mechanism by which these compounds inhibit FPTase has been investigated. The biochemical behavior of these compounds suggests that they bind to FPTase at the site usually occupied by the protein substrate. In experiments in cell culture, the methyl ester prodrugs of these inhibitors are cell permeant and potently inhibit the posttranslational modification of H-Ras protein. Additionally, these molecules revert the phenotype of ras transformed cells as evidenced by their ability to slow the growth of ras transformed cell lines in soft agar. One of the inhibitors, as its methyl prodrug, was evaluated in two in vivo models of tumor growth. The compound selectively inhibited the growth of tumors derived from H-ras transformed cells, in nude mice, and caused the regression of preexisting tumors in an H-ras transgenic animal model.

Intracellular recordings from isolated salamander olfactory receptor neurons.

Isolated receptor cells were obtained by enzymatic dissociation of nasal sacs from the land-phase tiger salamander. The isolated cells have an ovoid soma, a dendrite of variable length which terminates in a cilia-bearing knob and an axon, also of variable length. Intracellular recordings were obtained using patch pipettes. Good recordings were characterized by resting potentials of -40 mV, high input impedance and the presence of fast overshooting action potentials upon depolarization or rebound excitation. With one cell, chemical stimulation evoked large depolarizations which produced action potentials. The reversal potential of this response was +2.7 mV. The results show that these cells can be dissociated for patch recordings, and they support previous studies indicating that transduction of olfactory stimuli leads to a depolarization of vertebrate olfactory receptor neurons.

A physiological and biochemical model for digestion in the ectoparasitic mite, Psoroptes ovis (Acari: Psoroptidae).

Mites are an important group of arthropod pests affecting crops, animals and humans. Despite this, detailed physiological studies on these organisms remain sparse due largely to their small size. Unifying models are required to draw together the diverse information from studies on different groups and species. This paper describes a model for digestion in the parasitic mite, Psoroptes ovis, the causative agent of psoroptic mange or sheep scab disease. The limited information about this species is supplemented with data from other acarines, especially house dust mites and ticks. We review the range of enzymes and allergens found in mites and consider their possible roles in digestion in mites, generally and in particular, P. ovis. Histological studies, enzyme biochemistry and molecular biology and experimental evidence suggest that P. ovis utilises a digestive system reliant upon acid peptidases functioning in a largely intracellular environment. The actions of the digestive enzymes are supplemented by the involvement of bacteria as potential direct and indirect sources of nutrition. It is possible that some extra-corporeal digestion also takes place. The interaction of bacteria and digestive enzymes on the skin surface of the sheep may be responsible for the excessive pathological reactions evident in clinical sheep scab.

3,8-Diazabicyclo[3.2.1]octan-2-one peptide mimetics: synthesis of a conformationally restricted inhibitor of farnesyltransferase.

A new synthesis of the 3,8-diazabicyclo[3.2.1]octan-2-one framework is described. Transannular enolate alkylation of piperazinone derivatives provides a flexible route to highly constrained bicyclic peptidomimetic synthons with substitution at the Calpha position. The chemistry was used to produce a conformationally constrained farnesyltransferase inhibitor, which aided the elucidation of enzyme-bound conformation.

Safety of the insect repellent N,N-diethyl-M-toluamide (DEET) in pregnancy.

The safety of daily application of N, N-diethyl-m-toluamide (DEET) (1.7 g of DEET/day) in the second and third trimesters of pregnancy was assessed as part of a double-blind, randomized, therapeutic trial of insect repellents for the prevention of malaria in pregnancy (n = 897). No adverse neurologic, gastrointestinal, or dermatologic effects were observed for women who applied a median total dose of 214.2 g of DEET per pregnancy (range = 0-345.1 g). DEET crossed the placenta and was detected in 8% (95% confidence interval = 2.6-18.2) of cord blood samples from a randomly selected subgroup of DEET users (n = 50). No adverse effects on survival, growth, or development at birth, or at one year, were found. This is the first study to document the safety of DEET applied regularly in the second and third trimesters of pregnancy. The results suggest that the risk of DEET accumulating in the fetus is low and that DEET is safe to use in later pregnancy.

Intracellular potentials of salamander mitral/tufted neurons in response to odor stimulation.

Intracellular responses of salamander mitral/tufted neurons to defined odor pulses are described. Responses to odor stimulation, which generally are more complex than responses to olfactory nerve or tract electrical stimulation, show periods of depolarization and hyperpolarization that are influenced by odor concentration and quality. The way these periods coincide with different types of spike patterns in individual cells supports the hypothesis that the temporal patterning of odor responses is generated by differential activation of bulbar circuits.

Ultrastructural identification of synapses between mitral/tufted cell dendrites.

Asymmetrical, type 1 synapses between mitral and/or tufted (M/T) cell dendrites were observed in the glomerular layer (GL) and juxtaglomerular external plexiform layer (EPL) of salamander olfactory bulb sections. The dendrites had electron-lucent cytoplasm containing regularly-arrayed microtubules and spherical translucent vesicles. The vesicles were clustered against a thin pre-synaptic density that was aligned with a 17-20 nm-wide synaptic cleft and a thicker post-synaptic density. These dendrodendritic synapses could be a source of the delayed, prolonged excitation that originates from the GL/EPL. During spatiotemporal encoding of odor stimuli, they could amplify or synchronize M/T cell responses.

Processing of olfactory information at three neuronal levels in the spiny lobster.

Odor quality coding was analyzed at three neuronal levels, receptor cells and two levels of chemosensory interneurons, in the olfactory system of the spiny lobster Panulirus argus . Responses to three of the most stimulatory compounds for this animal - taurine, glutamate and betaine - were recorded at each level in order to compare basic neuronal response properties, single cell and population response spectra, and across-neuron patterns. Mean response specificity increased for cells at each successive neuronal level. The increase in breadth of tuning between receptor cells and low-order interneurons was paralleled by an increase in interstimulus across-neuron correlations. However, in high-order interneurons, there was a relative decline in across-neuron correlations, indicating that the more broadly-tuned high-order interneurons are better able to discriminate between any two compounds than are the more narrowly-tuned low-order interneurons. Although stimulus quality appears to be coded by interneurons as an across-fiber pattern, the fact that some low-order and high-order interneurons retained the narrow specificity of receptor cells suggests that labeled lines may have an important function in coding throughout the olfactory pathway of the spiny lobster.

Immunoreactivity for apolipoprotein A-IV in tanycytes and astrocytes of rat brain.

Immunoperoxidase and immunofluorescence procedures were used to visualize polyclonal antiserum binding to apolipoprotein (apo) A-IV in rat brain. With both methods, tanycytes and astrocytes were labeled throughout both white and gray matter. Within the cells, the labeling was granular and it was confined to the perinuclear zone and proximal regions of the processes. The labeling was abolished by absorption of the primary antiserum with purified apo A-IV but not by absorption with apo E. These results suggest either that apo A-IV is synthesized by astrocytes, or that apolipoprotein that is synthesized in the small intestine or liver is selectively taken up and stored by the astrocytes.