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Tangential-flow ultrafiltration was optimized for the recovery of Escherichia coli, Enterococcus faecalis, Clostridium perfringens spores, bacteriophages MS2 and PRD1, murine norovirus, and poliovirus seeded into 100-liter surface water (SW) and drinking water (DW) samples. SW and DW collected from two drinking water treatment plants were then evaluated for human enteric viruses. [ABSTRACT FROM AUTHOR]

Tangential flow ultrafiltration (UF) was used to concentrate and recover bacterial indicators and enteric viruses from 100 liters of groundwater (GW; n = 10) and surface water (SW; n = 11) samples collected in Lower Yakima Valley, WA. Human and bovine enteric viruses were analyzed in SW and GW concentrates by real-time PCR by using integrated inhibition detection. [ABSTRACT FROM AUTHOR]

Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (n = 9) and groundwater samples (n = 6). Viral persistence was monitored at 25°C and 4°C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25°C (0.18 and 0.09 log10/day for FCV, 0.13 and 0.10 log10/day for PV, 0.12 and 0.06 log10/day for MS2, and 0.09 and 0.05 log10/day for MNV) but not significant at 4°C. According to a multiple linear regression model, the NV NA reduction rates (0.04 ± 0.01 log10/day) were not significantly different from the NA reduction rates of MS2 (0.05 ± 0.03 log10/day) and MNV (0.04 ± 0.03 log10/day) and were significantly different from those of FCV (0.08 ± 0.03 log10/day) and PV (0.09 ± 0.03 log10/day) at 25°C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS) represents a rapid technique for the identification of microbial monocultures, and its adaptation to the detection of pathogens in real-world samples is a public health and homeland security priority. Norovirus, a leading cause of gastroenteritis in the world, is difficult to monitor because it cannot be cultured outside the human body. The detection of norovirus capsid protein was explored using three common MS-based methods: scanning of intact proteins, peptide mass fingerprinting, and peptide sequencing. Detection of intact target protein was limited by poor selectivity and sensitivity. Detection of up to 16 target peptides by peptide mass fingerprinting allowed for the reproducible and confident (P < 0.05) detection of the 56-kDa norovirus capsid protein in the range of 0.1 × 10-12 to 50 × 10-12 mol in authentic standards of recombinant norovirus virus-like particles (VLPs). To explore assay performance in complex matrixes, a non-gel-based, rapid method (2 to 3 h) for virus extraction from human stool was evaluated (72% ± 12% recovery), and additional analyses were performed on norovirus-free stool extracts fortified with VLPs. Whereas peptide mass fingerprinting was rendered impractical by sample interferences, peptide sequencing using nanospray tandem MS facilitated unambiguous identification of >250 fmol of capsid protein in stool extracts. This is the first report on MS-based detection of norovirus, accomplished by using structurally identical, noninfective VLPs at clinically relevant concentrations. It represents an important milestone in the development of assays for surveillance of this category B bioterrorism agent. [ABSTRACT FROM AUTHOR]

Infections caused by Toxoplasma gondii are widely prevalent in animals and humans throughout the world. In the United States, an estimated 23% of adolescents and adults have laboratory evidence of T. gondii infection. T. gondii has been identified as a major opportunistic pathogen in immunocompromised individuals, in whom it can cause life-threatening disease. Water contaminated with feces from domestic cats or other felids may be an important source of human exposure to T. gondii oocysts. Because of the lack of information regarding the prevalence of T. gondii in surface waters, there is a clear need for a rapid, sensitive method to detect T. gondii from water. Currently available animal models and cell culture methods are time-consuming, expensive, and labor-intensive, requiring days or weeks for results to be obtained. Detection of T. gondii nucleic acid by PCR has become the preferred method. We have developed a PCR amplification and detection method for T. gondii oocyst nucleic acid that incorporates the use of hot-start amplification to reduce nonspecific primer annealing, uracii-N-glycosylase to prevent false-positive results due to carryover contamination, an internal standard control to identify false-negative results due to inadequate removal of sample inhibition, and PCR product oligoprobe confirmation using a nonradioactive DNA hybridization immunoassay. This method can provide positive, confirmed results in less than 1 day. Fewer than 50 oocysts can be detected following recovery of oocyst DNA. Development of a T. gondii oocyst PCR detection method will provide a useful technique to estimate the levels of T. gondii oocysts present in surface waters. [ABSTRACT FROM AUTHOR]

Reports on the development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of 'norwalk-like' viruses and hepatitis A virus in stool and shellfish. Main cause of outbreaks of food- and waterborne gastroenteritis; Principles of the developed method; General applicability of the developed method in the detection of viruses.

Focuses on methods for the detection of Norwalk-like viruses (NLV) and hepatitis A virus (HAV) in delicatessen foods. Isolation and purification of NLV from deli meats; Detection of genogroup II NLV in ham using polymerase- and capsid-specific primers and probes; Similarity of capsid sequences from stool and ham samples.

To examine the long-term infectivity of human norovirus in water, 13 study subjects were challenged at different time points with groundwater spiked with the prototype human norovirus, Norwalk virus. Norwalk virus spiked in groundwater remained infectious after storage at room temperature in the dark for 61 days (the last time point tested). The Norwalk virus-seeded groundwater was stored for 1,266 days and analyzed, after RNase treatment, by reverse transcription-quantitative PCR (RT-qPCR) to detect Norwalk virus RNA contained within intact capsids. Norwalk virus RNA within intact capsids was detected in groundwater for 1,266 days, with no significant log10 reduction throughout 427 days and a significant 1.10-log10 reduction by day 1266. Purified Norwalk virus RNA (extracted from Norwalk virus virions) persisted for 14 days in groundwater, tap water, and reagent-grade water. This study demonstrates that Norwalk virus in groundwater can remain detectable for over 3 years and can remain infectious for at least 61 days. [ABSTRACT FROM AUTHOR]

Crassostrea ariakensis oysters are under review for introduction into the Chesapeake Bay. However, the human health implications of the introduction have not been fully addressed. This study evaluated rates of bioaccumulation, retention, and depuration of viruses by Crassostrea virginica and C. ariakensis when the two oyster species were maintained in separate tanks containing synthetic seawater of various salinities (8, 12, or 20 ppt). Oyster bioaccumulation tanks were seeded with 103 PFU/ml of hepatitis A virus (HAV), poliovirus, male-specific bacteriophage (MS2), and murine norovirus 1 (MNV-1) and 103 PCR units/mI of human norovirus (NoV). After 24 h, depuration commenced as oysters (n = 255) were placed in pathogen-free seawater under continuous filtration. Oysters (n = 6) were sampled weekly for 1 month from each tank. Viral RNA was recovered using a modified proteinase K, guanidine, and glassmilk method and analyzed by quantitative reverse transcription-PCR. The odds of C. ariakensis oysters harboring NoV, MNV-1, or HAV were statistically greater than the odds of C. virginica oysters harboring the same viruses (MNV-1 odds ratio [OR], 4.5; P = 0.01; NoV OR, 8.4; P < 0.001; HAV OR, 11.4; P < 0.001). Unlike C. virginica, C. ariakensis bioaccumulated and retained NoV, MNV-1, and HAY for 1 month at all salinities. Additionally, the odds of an oyster testing positive for NoV was 25.5 times greater (P < 0.001) when the oyster also tested positive for MNV-1. This research helps assess the threat of C. ariakensis as a vehicle for viral pathogens due to the consumption of raw oysters and validates the role for MNV-1 as a surrogate for NoV. [ABSTRACT FROM AUTHOR]

Human noroviruses (hNoVs) are a known public health concern associated with the consumption of leafy green vegetables. While a number of studies have investigated pathogen reduction on the surfaces of leafy greens during the postharvest washing process, there remains a paucity of data on the level of treatment needed to inactivate viruses in the wash water, which is critical for preventing cross-contamination. The objective of this study was to quantify the susceptibility of hNoV genotype I (GI), hNoV GII, murine norovirus (MNV), and bacteriophage MS2 to free chlorine in whole leaf, chopped romaine, and shredded iceberg lettuce industrial leafy green wash waters, each sampled three times over a 4-month period. A suite of kinetic inactivation models was fit to the viral reduction data to aid in quantification of concentration-time (CT) values. Results indicate that 3-log10 infectivity reduction was achieved at CT values of less than 0.2 mg · min/liter for MNV and 2.5 mg · min/liter for MS2 in all wash water types. CT values for 2-log10 molecular reduction of hNoV GI in whole leaf and chopped romaine wash waters were 1.5 and 0.9 mg · min/liter, respectively. For hNoV GII, CT values were 13.0 and 7.5 mg · min/liter, respectively. In shredded iceberg wash water, 3-log10 molecular reduction was not observed for any virus over the time course of experiments. These findings demonstrate that noroviruses may exhibit genogroup-dependent resistance to free chlorine and emphasize the importance of distinguishing between genogroups in hNoV persistence studies. IMPORTANCE Postharvest washing of millions of pounds of leafy greens is performed daily in industrial processing facilities with the intention of removing dirt, debris, and pathogenic microorganisms prior to packaging. Modest inactivation of pathogenic microorganisms (less than 2 log10) is known to occur on the surfaces of leafy greens during washing. Therefore, the primary purpose of the sanitizing agent is to maintain microbial quality of postharvest processing water in order to limit cross-contamination. This study modeled viral inactivation data and quantified the free-chlorine CT values that processing facilities must meet in order to achieve the desired level of hNoV GI and GII reduction. Disinfection experiments were conducted in industrial leafy green wash water collected from a full-scale fresh produce processing facility in the United States, and hNoV GI and GII results were compared with surrogate molecular and infectivity data. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus naturally occurs in brackish and marine waters and is one of the leading causes of seafood-borne illness. Previous work studying the ecology of V. parahaemolyticus has often been limited in geographic extent and lacked a full range of environmental measures. This study used a unique large data set of surface water samples in the Chesapeake Bay (n = 1,385) collected from 148 monitoring stations from 2007 to 2010. Water was analyzed for more than 20 environmental parameters, with additional meteorological and surrounding land use data. The V. parahaemolyticus-specific genetic markers thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) were assayed using quantitative PCR (qPCR), and interval-censored regression models with nonlinear effects were estimated to account for limits of detection and quantitation. tlh was detected in 19.6% of water samples; tdh or trh markers were not detected. The results confirmed previously reported positive associations for V. parahaemolyticus abundance with temperature and turbidity and negative associations with high salinity (>10 to 23‰). Furthermore, the salinity relationship was determined to be a function of both low temperature and turbidity, with an increase of either nullifying the high salinity effect. Associations with dissolved oxygen and phosphate also appeared stronger when samples were taken near human developments. A renewed focus on the V. parahaemolyticus ecological paradigm is warranted to protect public health. IMPORTANCE Vibrio parahaemolyticus is one of the leading causes of seafood-borne illness in the United States and across the globe. Exposure is often through consuming raw or undercooked shellfish. Given the natural presence of the bacterium in the marine environment, an improved understanding of its environmental determinants is necessary for future preventative measures. This analysis of environmental Vibrio parahaemolyticus is one of only a few that utilize a large data set measured over a wide geographic and temporal range. The analysis also includes a large number of environmental parameters for Vibrio modeling, many of which have previously only been tested sporadically, and some of which have not been considered before. The results of the analysis revealed previously unknown relationships between salinity, turbidity, and temperature that provide significant insight into the abundance and persistence of V. parahaemolyticus bacterium in the environment. This information will be essential for developing environmental forecast models for the bacterium. [ABSTRACT FROM AUTHOR]

Manure spills into streams are relatively frequent, but no studies have characterized stream contamination with zoonotic and veterinary pathogens, or fecal chemicals, following a spill. We tested stream water and sediment over 25 days and downstream for 7.6 km for the following: fecal indicator bacteria (FIB), the fecal indicator chemicals cholesterol and coprostanol, 20 genes for zoonotic and swine-specific bacterial pathogens by presence/absence PCR for viable cells, one swine-specific Escherichia coli toxin gene (STII gene) by quantitative PCR (qPCR), and nine human and animal viruses by qPCR or reverse transcription-qPCR. Twelve days postspill, and 4.2 km downstream, water concentrations of FIB, cholesterol, and coprostanol were 1 to 2 orders of magnitude greater than those detected before, or above, the spill, and genes indicating viable zoonotic or swine-infectious Escherichia coli were detected in water or sediment. STII gene levels increased from undetectable before or above the spill to 105 copies/ 100 ml of water 12 days postspill. Thirteen of 14 water (8/9 sediment) samples had viable STII-carrying cells postspill. Eighteen days postspill, porcine adenovirus and teschovirus were detected 5.6 km downstream. FIB concentrations (per gram [wet weight]) in sediment were greater than in water, and sediment was a continuous reservoir of genes and chemicals postspill. Constituent concentrations were much lower, and detections less frequent, in a runoff event (200 days postspill) following manure application, although the swine-associated STII and stx2e genes were detected. Manure spills are an underappreciated pathway for livestock-derived contaminants to enter streams, with persistent environmental outcomes and the potential for human and veterinary health consequences. [ABSTRACT FROM AUTHOR]

The increased awareness of the role of environmental matrices in enteric disease transmission has resulted in the need for rapid, field-based methods for fecal indicator bacteria and pathogen detection. Evidence of the specificity of β-glucuronidase-based assays for detection of Escherichia coli from environmental matrices relevant to enteric pathogen transmission in developing countries, such as hands, soils, and surfaces, is limited. In this study, we quantify the false-positive rate of a β-glucuronidasebased E. coli detection assay (Colilert) for two environmental reservoirs in Bangladeshi households (hands and soils) and three fecal composite sources (cattle, chicken, and humans). We investigate whether or not the isolation source of E. coli influences phenotypic and genotypic characteristics. Phenotypic characteristics include results of biochemical assays provided by the API- 20E test; genotypic characteristics include the Clermont phylogroup and the presence of enteric and/or environmental indicator genes sfmH, rfaI, and fucK. Our findings demonstrate no statistically significant difference in the false-positive rate of Colilert for environmental compared to enteric samples. E. coli isolates from all source types are genetically diverse, representing six of the seven phylogroups, and there is no difference in relative frequency of phylogroups between enteric and environmental samples. We conclude that Colilert, and likely other β-glucuronidase-based assays, is appropriate for detection of E. coli on hands and in soils with low false-positive rates. Furthermore, E. coli isolated from hands and soils in Bangladeshi households are diverse and indistinguishable from cattle, chicken, and human fecal isolates, using traditional biochemical assays and phylogrouping. [ABSTRACT FROM AUTHOR]

The introduction of nonnative oysters (i.e., Crassostrea ariakensis) into the Chesapeake Bay has been proposed as necessary for the restoration of the oyster industry; however, nothing is known about the public health risks related to contamination of these oysters with human pathogens. Commercial market-size C. ariakensis triploids were maintained in large marine tanks with water of low (8-ppt), medium (12-ppt), and high (20-ppt) salinities spiked with 1.0 × 105 transmissive stages of the following human pathogens: Cryptosporidium parvum oocysts, Giardia lamblia cysts, and microsporidian spores (i.e., Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi). Viable oocysts and spores were still detected in oysters on day 33 post-water inoculation (pwi), and cysts were detected on day 14 pwi. The recovery, bioaccumulation, depuration, and inactivation rates of human waterborne pathogens by C. ariakensis triploids were driven by salinity and were optimal in medium- and high-salinity water. The concentration of human pathogens from ambient water by C. ariakensis and the retention of these pathogens without (or with minimal) inactivation and a very low depuration rate provide evidence that these oysters may present a public health threat upon entering the human food chain, if harvested from polluted water. This conclusion is reinforced by the concentration of waterborne pathogens used in the present study, which was representative of levels of infectious agents in surface waters, including the Chesapeake Bay. Aquacultures of nonnative oysters in the Chesapeake Bay will provide excellent ecological services in regard to efficient cleaning of human-infectious agents from the estuarine waters. [ABSTRACT FROM AUTHOR]

Organoarsenicals are commonly used for growth promotion in U.S. poultry production. Susceptibilities to arsenite, arsenate, and the organoarsenical roxarsone were measured in 251 Campylobacter isolates from conventional and antimicrobial-free retail poultry products. Isolates from conventional poultry products had significantly higher roxarsone MICs (z = 8.22; P < 0.0001). [ABSTRACT FROM AUTHOR]

This study demonstrated that a person with 30 min of occupational or nonoccupational exposure to urban feral pigeons, such as exposure through the cleaning of surfaces contaminated with pigeon excrement, could inhale approximately 3.5 × 103 Enterocytozoon bieneusi spores and that 1.3 × 103 spores could be inhaled by a nearby person. [ABSTRACT FROM AUTHOR]