Evaluation of Different Methods for Detecting Male Specific Coliphages from Marine Water at Doheny Beach, CA

Male-specific (F+) coliphages, primarily F+ RNA coliphages, have been proposed as candidate indicators of enteric viruses in water. The purpose of the this study was to evaluate the use of F+ coliphages as viral indicators of fecal contamination in marine waters using: (1) the Coliphage Latex Agglutination and Typing (CLAT) immunoassay, a rapid and novel coliphage detection and typing method, (2) a rapid (5-hr) liquid culture enrichment, and (3) a standard overnight liquid culture enrichment method, EPA Method 1601, at Doheny Beach, CA. This study analyzed 75 1 L marine water samples following rapid enrichment and 101 samples following overnight enrichment. For both methods, samples were collected in 2007 and 2008. The study was continued for the summer of 2008 to better document the performance of modifications of the rapid coliphages methods that were intended to overcome deficiencies of the method identified in 2007. For samples collected in 2007 using the rapid-CLAT and the overnight-CLAT assay 3/75(4.00%) and 4/101(3.96%) samples were positive for F+ coliphages respectively. Higher detection rates were found by using rapid and overnight enrichment spot plating with 16/75(21.3%) and 27/101(26.7%), respectively. We tested the lysis zones of the spot plates for the presence of F+ coliphage with the CLAT and found that 16/101(15.8%) were positive for rapid and 23/101(22.7%) for overnight enrichments, respectively, both of which were significant increases in positivity (p<0.05). We analyzed the effect salinity has on the enrichment and found that by reducing the salt concentration of the seawater samples by adding 1-liter of salt-free water, the growth of E. coli and the propagation of coliphages was improved. E. coli growth during the rapid (5-hr) enrichment improved from 9.0x10^8 to 1.0x1O^9 cfu/mL and coliphage propagation improved from 2.3x10^5 to 1.0x10^6 pfu/mL. This dilution of seawater was used for the analysis of 75 samples in 2008. Using the rapid-CLAT and the overnight-CLAT assay 16/75 (21.3%) and 24/101(23.7%) samples were positive for F+ coliphages respectively in 2008. Following rapid and overnight enrichment spot plating we found 37/75(49.3%) and 76/101(75.2%) F+ coliphage-positive samples, respectively, which was significantly different by Fisher Exact Test analysis (p<005). The results of this study suggest that the CLAT assay needs further improvement to (1) increase coliphage virus detection in seawater enrichments or(2) reduce to a lower coliphage concentration the lower detection limit of the agglutination immunoassay as approaches to achieving maximum F+ coliphage detection.Master of Science in Public Healt

Particulate matter neurotoxicity in culture is size-dependent

Exposure to particulate matter (PM) air pollution produces inflammatory damage to the cardiopulmonary system. This toxicity appears to be inversely related to the size of the PM particles, with the ultrafine particle being more inflammatory than larger sizes. Exposure to PM has more recently been associated with neurotoxicity. This study examines if the size-dependent toxicity reported in cardiopulmonary systems also occurs in neural targets. For this study, PM ambient air was collected over a 2 week period from Sterling Forest State Park (Tuxedo, New York) and its particulates sized as Accumulation Mode, Fine (AMF) (>0.18-1μm) or Ultrafine (UF) (12.5μg/ml) but was only significant at the highest concentration of AMF (50μg/ml). To examine if PM size-dependent neurotoxicity was retained in the presence of other cell types, dissociated brain cultures of embryonic rat striatum were exposed to AMF (80μg/ml) or UF (8.0μg/ml). After 24h exposure, a significant increase of reactive nitrogen species (nitrite) and morphology suggestive of apoptosis occurred in both treatment groups. However, morphometric analysis of neuron specific enolase staining indicated that only the UF exposure produced significant neuronal loss, relative to controls. Together, these data suggest that the inverse relationship between size and toxicity reported in cardiopulmonary systems occurs in cultures of isolated dopaminergic neurons and in primary cultures of the rat striatum

Nanosize Titanium Dioxide Stimulates Reactive Oxygen Species in Brain Microglia and Damages Neurons in Vitro

BackgroundTitanium dioxide is a widely used nanomaterial whose photo-reactivity suggests that it could damage biological targets (e.g., brain) through oxidative stress (OS).ObjectivesBrain cultures of immortalized mouse microglia (BV2), rat dopaminergic (DA) neurons (N27), and primary cultures of embryonic rat striatum, were exposed to Degussa P25, a commercially available TiO2 nanomaterial. Physical properties of P25 were measured under conditions that paralleled biological measures.FindingsP25 rapidly aggregated in physiological buffer (800–1,900 nm; 25°C) and exposure media (~ 330 nm; 37°C), and maintained a negative zeta potential in both buffer (–12.2 ± 1.6 mV) and media (–9.1 ± 1.2 mV). BV2 microglia exposed to P25 (2.5–120 ppm) responded with an immediate and prolonged release of reactive oxygen species (ROS). Hoechst nuclear stain was reduced after 24-hr (≥100 ppm) and 48-hr (≥2.5 ppm) exposure. Microarray analysis on P25-exposed BV2 microglia indicated up-regulation of inflammatory, apoptotic, and cell cycling pathways and down-regulation of energy metabolism. P25 (2.5–120 ppm) stimulated increases of intracellular ATP and caspase 3/7 activity in isolated N27 neurons (24–48 hr) but did not produce cytotoxicity after 72-hr exposure. Primary cultures of rat striatum exposed to P25 (5 ppm) showed a reduction of immunohistochemically stained neurons and microscopic evidence of neuronal apoptosis after 6-hr exposure. These findings indicate that P25 stimulates ROS in BV2 microglia and is nontoxic to isolated N27 neurons. However, P25 rapidly damages neurons at low concentrations in complex brain cultures, plausibly though microglial generated ROS

Comparison of methods for the detection of coliphages in recreational water at two California, United States beaches

Methods for detection of two fecal indicator viruses, F+ and somatic coliphages, were evaluated for application to recreational marine water. Marine water samples were collected during the summer of 2007 in Southern California, United States from transects along Avalon Beach (n = 186 samples) and Doheny Beach (n = 101 samples). Coliphage detection methods included EPA method 1601 – two-step enrichment (ENR), EPA method 1602 – single agar layer (SAL), and variations of ENR. Variations included comparison of two incubation times (overnight and 5-h incubation) and two final detection steps (lysis zone assay and a rapid latex agglutination assay). A greater number of samples were positive for somatic and F+ coliphages by ENR than by SAL (p \u3c 0.01). The standard ENR with overnight incubation and detection by lysis zone assay was the most sensitive method for the detection of F+ and somatic coliphages from marine water, although the method takes up to three days to obtain results. A rapid 5-h enrichment version of ENR also performed well, with more positive samples than SAL, and could be performed in roughly 24 h. Latex agglutination-based detection methods require the least amount of time to perform, although the sensitivity was less than lysis zone-based detection methods. Rapid culture-based enrichment of coliphages in marine water may be possible by further optimizing culture-based methods for saline water conditions to generate higher viral titers than currently available, as well as increasing the sensitivity of latex agglutination detection methods