Kellogg J. Schwab

Evaluation of Murine Norovirus, Feline Calicivirus, Poliovirus, and MS2 as Surrogates for Human Norovirus in a Model of Viral Persistence in Surface Water and Groundwater

ABSTRACT 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.

A Norovirus Outbreak at a Long-Term-Care Facility: The Role of Environmental Surface Contamination

BACKGROUND The role of environmental surface contamination in the propagation of norovirus outbreaks is unclear. An outbreak of acute gastroenteritis was reported among residents of a 240-bed veterans long-term-care facility. OBJECTIVES To identify the likely mode of transmission, to characterize risk factors for illness, and to evaluate for environmental contamination in this norovirus outbreak. METHODS An outbreak investigation was conducted to identify risk factors for illness among residents and employees. Stool and vomitus samples were tested for norovirus by reverse transcription polymerase chain reaction (RT-PCR). Fourteen days after outbreak detection, ongoing cases among the residents prompted environmental surface testing for norovirus by RT-PCR. RESULTS One hundred twenty-seven (52%) of 246 residents and 84 (46%) of 181 surveyed employees had gastroenteritis. Case-residents did not differ from non-case-residents by comorbidities, diet, room type, or level of mobility. Index cases were among the nursing staff. Eight of 11 resident stool or vomitus samples tested positive for genogroup II norovirus. The all-cause mortality rate during the month of the outbreak peak was significantly higher than the expected rate. Environmental surface swabs from case-resident rooms, a dining room table, and an elevator button used only by employees were positive for norovirus. Environmental and clinical norovirus sequences were identical. CONCLUSION Extensive contamination of environmental surfaces may play a role in prolonged norovirus outbreaks and should be addressed in control interventions.

Norovirus Infectivity in Humans and Persistence in Water

ABSTRACT 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. (ClinicalTrials.gov identifier NCT00313404.)

Development of Methods To Detect “Norwalk-Like Viruses” (NLVs) and Hepatitis A Virus in Delicatessen Foods: Application to a Food-Borne NLV Outbreak

ABSTRACT “Norwalk-like viruses” (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 102 to 103 viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.

Antibiotic-Resistant Enterococci and Fecal Indicators in Surface Water and Groundwater Impacted by a Concentrated Swine Feeding Operation

Background The nontherapeutic use of antibiotics in swine feed can select for antibiotic resistance in swine enteric bacteria. Leaking swine waste storage pits and the land-application of swine manure can result in the dispersion of resistant bacteria to water sources. However, there are few data comparing levels of resistant bacteria in swine manure–impacted water sources versus unaffected sources. Objectives The goal of this study was to analyze surface water and groundwater situated up and down gradient from a swine facility for antibiotic-resistant enterococci and other fecal indicators. Methods Surface water and groundwater samples (n = 28) were collected up and down gradient from a swine facility from 2002 to 2004. Fecal indicators were isolated by membrane filtration, and enterococci (n = 200) were tested for susceptibility to erythromycin, tetracycline, clindamycin, virginiamycin, and vancomycin. Results Median concentrations of enterococci, fecal coliforms, and Escherichia coli were 4- to 33-fold higher in down-gradient versus up-gradient surface water and groundwater. We observed higher minimal inhibitory concentrations for four antibiotics in enterococci isolated from down-gradient versus up-gradient surface water and groundwater. Elevated percentages of erythromycin- (p = 0.02) and tetracycline-resistant (p = 0.06) enterococci were detected in down-gradient surface waters, and higher percentages of tetracycline- (p = 0.07) and clindamycin-resistant (p < 0.001) enterococci were detected in down-gradient groundwater. Conclusions We detected elevated levels of fecal indicators and antibiotic-resistant enterococci in water sources situated down gradient from a swine facility compared with up-gradient sources. These findings provide additional evidence that water contaminated with swine manure could contribute to the spread of antibiotic resistance.

Airborne Multidrug-Resistant Bacteria Isolated from a Concentrated Swine Feeding Operation

The use of nontherapeutic levels of antibiotics in swine production can select for antibiotic resistance in commensal and pathogenic bacteria in swine. As a result, retail pork products, as well as surface and groundwaters contaminated with swine waste, have been shown to be sources of human exposure to antibiotic-resistant bacteria. However, it is unclear whether the air within swine operations also serves as a source of exposure to antibiotic-resistant bacterial pathogens. To investigate this issue, we sampled the air within a concentrated swine feeding operation with an all-glass impinger. Samples were analyzed using a method for the isolation of Enterococcus. A total of 137 presumptive Enterococcus isolates were identified to species level using standard biochemical tests and analyzed for resistance to erythromycin, clindamycin, virginiamycin, tetracycline, and vancomycin using the agar dilution method. Thirty-four percent of the isolates were confirmed as Enterococcus, 32% were identified as coagulase-negative staphylococci, and 33% were identified as viridans group streptococci. Regardless of bacterial species, 98% of the isolates expressed high-level resistance to at least two antibiotics commonly used in swine production. None of the isolates were resistant to vancomycin, an antibiotic that has never been approved for use in livestock in the United States. In conclusion, high-level multidrug-resistant Enterococcus, coagulase-negative staphylococci, and viridans group streptococci were detected in the air of a concentrated swine feeding operation. These findings suggest that the inhalation of air from these facilities may serve as an exposure pathway for the transfer of multidrug-resistant bacterial pathogens from swine to humans.

Effects of Solution Chemistry on the Adsorption of Ibuprofen and Triclosan onto Carbon Nanotubes

Single-walled carbon nanotubes (SWCNTs), multiwalled carbon nanotubes (MWCNTs), and oxidized MWCNTs (O-MWCNTs) were studied for the adsorption of ibuprofen (IBU) and triclosan (TCS) as representative types of pharmaceutical and personal care products (PPCPs) under different chemical solution conditions. A good fitting of sorption isotherms was obtained using a Polanyi-Manes model (PMM). IBU and TCS sorption was stronger for SWCNTs than for MWCNTs due to higher specific surface area. The high oxygen content of O-MWCNT further depressed PPCP sorption. The sorption capacity of PPCPs was found to be pH-dependent, and more adsorption was observed at pHs below their pK(a) values. Ionic strength was also found to substantially affect TCS adsorption, with higher adsorption capacity observed for TCS at lower ionic strength. In the presence of a reference aquatic fulvic acid (FA), sorption of IBU and TCS was reduced due to the competitive sorption of FA on carbon nanotubes (CNTs). Sorption isotherm results with SWCNTs, MWCNTs and O-MWCNTs confirmed that the surface chemistry of CNTs, the chemical properties of PPCPs, and aqueous solution chemistry (pH, ionic strength, fulvic acid) all play an important role in PPCP adsorption onto CNTs.

Bioaccumulation, Retention, and Depuration of Enteric Viruses by Crassostrea virginica and Crassostrea ariakensis Oysters

ABSTRACT 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/ml 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 HAV 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.

The role of birds in dissemination of human waterborne enteropathogens

Cryptosporidiosis, giardiasis and microsporidiosis are serious human diseases of waterborne origin; their etiologic agents and a substantial fecal coliform load can enter surface, drinking and recreational water resources from aquatic birds. The aim of this article is to present interactions between waterfowl and these waters that imply a negative public health impact, reinforcing the need for either better water-quality indicators or for water monitoring specifically for Cryptosporidium, Giardia and microsporidia. Where justifiable, the presence of waterfowl should be supported; however, management of drinking and recreational water resources needs to be improved by incorporating effective protection measures for pathogens linked to these birds.

Fecal Contamination and Diarrheal Pathogens on Surfaces and in Soils among Tanzanian Households with and without Improved Sanitation

Little is known about the extent or pattern of environmental fecal contamination among households using low-cost, on-site sanitation facilities, or what role environmental contamination plays in the transmission of diarrheal disease. A microbial survey of fecal contamination and selected diarrheal pathogens in soil (n = 200), surface (n = 120), and produce samples (n = 24) was conducted in peri-urban Bagamoyo, Tanzania, among 20 households using private pit latrines. All samples were analyzed for E. coli and enterococci. A subset was analyzed for enterovirus, rotavirus, norovirus GI, norovirus GII, diarrheagenic E. coli, and general and human-specific Bacteroidales fecal markers using molecular methods. Soil collected from the house floor had significantly higher concentrations of E. coli and enterococci than soil collected from the latrine floor. There was no significant difference in fecal indicator bacteria levels between households using pit latrines with a concrete slab (improved sanitation) versus those without a slab. These findings imply that the presence of a concrete slab does not affect the level of fecal contamination in the household environment in this setting. Human Bacteroidales, pathogenic E. coli, enterovirus, and rotavirus genes were detected in soil samples, suggesting that soil should be given more attention as a transmission pathway of diarrheal illness in low-income countries.