Are existing bacterial indicators adequate for determining recreational water illness in waters impacted by nonpoint pollution?
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Environmental Exposure↗
Biomedical subjects
Kellogg J Schwab
Publications and source records attributed to Kellogg J Schwab.
Detection of norovirus capsid protein in authentic standards and in stool extracts by matrix-assisted laser desorption ionization and nanospray mass spectrometry.
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 x 10(-12) to 50 x 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 > or =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.
Amino Acid Sequence↗
Arsenic resistance in Campylobacter spp. isolated from retail poultry products.
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).
Animals↗
Recovery, bioaccumulation, and inactivation of human waterborne pathogens by the Chesapeake Bay nonnative oyster, Crassostrea ariakensis.
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 x 10(5) 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.
Animals↗
Assessment of an aerosol treatment to improve air quality in a swine concentrated animal feeding operation (CAFO).
Poor air quality within swine concentrated animal feeding operations (CAFOs) poses a threat to workers, the surrounding community, and farm production. Accordingly, the current study was conducted to evaluate a technology for reducing air pollution including particulate matter (PM), viable bacteria, and ammonia within such a facility. The technology consists of an acid-oil-alcohol aerosol applied daily. Its effectiveness was evaluated by comparing air quality from before to after treatment and between treated and untreated sides of a barn separated by an impervious partition. On the untreated side, air quality was typical for a swine CAFO, with mean PM2.5 of 0.28 mg/m3 and PM(TOT) of 1.5 mg/m3. The treatment yielded a reduction in PM concentration of 75-90% from before to after treatment. Effectiveness increased with time, application, and particle size (40% reduction for 1 microm and 90% for >10 microm). Airborne bacteria levels (total bacteria, Enterobacteriaceae, and gram-positive cocci) decreased one logarithmic unit after treatment. In contrast, treatment had no effect on ammonia concentrations. These findings demonstrate the effectiveness of an intervention in yielding exposure and emission reductions.
Acids↗
Riverbank filtration for control of microorganisms: results from field monitoring.
Microbial monitoring was conducted over a period of more than 1 year at three full-scale riverbank filtration (RBF) facilities, located in the United States along the Ohio, Missouri, and Wabash Rivers. Results of this study demonstrated the potential for RBF to provide substantial reductions in microorganism concentrations relative to the raw water sources. Cryptosporidium and Giardia were detected occasionally in the river waters but never in any of the well waters. Average concentrations and log reductions of Cryptosporidium and Giardia could not be accurately determined due to the low and variable concentrations in the river waters and the lack of detectable concentrations in the well waters. Average concentrations of aerobic and anaerobic spore-forming bacteria, which have both been proposed as potential surrogates for the protozoans, were reduced at the three facilities by 0.8 to > 3.1 logs and 0.4 to > 4.9 logs, respectively. Average concentrations of male-specific and somatic bacteriophage were reduced by > 2.1 logs and 3.2 logs, respectively. Total coliforms were rarely detected in the well waters, with 5.5 and 6.1 log reductions in average concentrations at the two wells at one of the sites relative to the river water. Average turbidity reductions upon RBF at the three sites were between 2.2 and 3.3 logs. Turbidity and microbial concentrations in the river waters generally tracked the river discharge; a similar relationship between the well water concentrations and river discharge was not observed, due to the low, relatively constant well water turbidities and lack of a significant number of detections of microorganisms in the well waters. Further research is needed to better understand the relationships among transport of pathogens (e.g., Cryptosporidium, Giardia, viruses) and potential surrogate parameters (including bacterial spores and bacteriophage) during RBF and the effects of water and sediment characteristics on removal efficiency.
Environmental Monitoring↗
Use of green fluorescent protein-expressing Aspergillus fumigatus conidia to validate quantitative PCR analysis of air samples collected on filters.
This study used green fluorescent protein (GFP)-expressing Aspergillus fumigatus conidia to compare quantitative PCR (qPCR) enumeration with direct epifluorescent microscopic filter counts of conidia collected on filters in a test chamber. In separate experiments this study initially compared white versus fluorescent light microscopy for counting A. fumigatus conidia, then compared fluorescent microscopy counting of corresponding filter halves, and finally compared qPCR enumeration to counting by fluorescent light microscopy. The use of GFP-expressing conidia with epifluorescent microscopy yielded significantly higher conidia counts (p = 0.026, n = 41, mean of 4.1 conidia per counting field) and 40% faster counting times when compared to conventional counting using white light microscopy. GFP-expressing conidia were aerosolized in a test chamber and collected onto filters. Filters were divided in half and GFP-expressing conidia enumerated. There was no significant difference in the average conidia count per field between corresponding filter halves (p = 0.3, n = 9 filters, mean of 7.8 conidia per counting field). Thus, one filter half could be counted optically and would provide a reliable estimate of filter loading of the corresponding half, which could then be analyzed by qPCR. Filters (n = 38) loaded with GFP conidia in the aerosol chamber were divided in half and analyzed by either fluorescent microscopy or qPCR. The estimated filter loadings ranged from 15-30,000 conidia per filter. There was a linear relationship with a nearly 1:1 ratio between qPCR and direct microscopic estimates of filter loading (y = 1.06x + 404; R(2) = 0.91) showing that the outlined qPCR analysis method is in agreement with an external reference method and is reliable for enumerating A. fumigatus conidia collected on filters. The comparative data derived using GFP-expressing conidia confirmed that qPCR provides sensitive and accurate quantification of DNA from airborne conidia collected on filters.
Air Pollution, Indoor↗
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.
Adult↗
Prevalence of norovirus among visitors from the United States to Mexico and Guatemala who experience traveler's diarrhea.
Traveler's diarrhea (TD) is the most common infectious illness acquired by visitors to developing nations. The purpose of this study was to utilize molecular diagnostic techniques to determine the prevalence of norovirus (NoV) in TD occurring among visitors from the United States to Guatemala and Mexico. Stool samples (n = 54) were collected from 34 TD cases and analyzed for NoV by reverse transcription-PCR and oligoprobe confirmation. The overall prevalence of NoV was 65%. Interestingly, all NoV-positive stool samples were identified as genogroup I NoVs, and time spent at travel destinations was found to be an important factor in determining the frequency of infection (P = 0.003). Eleven NoV-positive stool samples also tested positive for enterotoxigenic Escherichia coli, indicating that dual infections with this leading bacterial cause of TD were very common. Results of this study suggest that NoV infection is a frequent occurrence among travelers to Mexico and Guatemala who experience episodes of TD. In addition, the simple molecular detection method utilized here will serve to facilitate more in-depth epidemiological studies of this emergent viral pathogen in travelers and other at-risk populations.
Blotting, Southern↗
Deficiencies in drinking water distribution systems in developing countries.
Rapidly growing populations and migration to urban areas in developing countries has resulted in a vital need for the establishment of centralized water systems to disseminate potable water to residents. Protected source water and modern, well-maintained drinking water treatment plants can provide water adequate for human consumption. However, ageing, stressed or poorly maintained distribution systems can cause the quality of piped drinking water to deteriorate below acceptable levels and pose serious health risks. This review will outline distribution system deficiencies in developing countries caused by: the failure to disinfect water or maintain a proper disinfection residual; low pipeline water pressure; intermittent service; excessive network leakages; corrosion of parts; inadequate sewage disposal; and inequitable pricing and usage of water. Through improved research, monitoring and surveillance, increased understanding of distribution system deficiencies may focus limited resources on key areas in an effort to improve public health and decrease global disease burden.
Developing Countries↗
Development of a method to detect and quantify Aspergillus fumigatus conidia by quantitative PCR for environmental air samples.
Exposure to Aspergillus fumigatus is linked with respiratory diseases such as asthma, invasive aspergillosis, hypersensitivity pneumonitis, and allergic bronchopulmonary aspergillosis. Molecular methods using quantitative PCR (qPCR) offer advantages over culture and optical methods for estimating human exposures to microbiological agents such as fungi. We describe an assay that uses lyticase to digest A. fumigatus conidia followed by TaqMan qPCR to quantify released DNA. This method will allow analysis of airborne A. fumigatus samples collected over extended time periods and provide a more representative assessment of chronic exposure. The method was optimized for environmental samples and incorporates: single tube sample preparation to reduce sample loss, maintain simplicity, and avoid contamination; hot start amplification to reduce non-specific primer/probe annealing; and uracil-N-glycosylase to prevent carryover contamination. An A. fumigatus internal standard was developed and used to detect PCR inhibitors potentially found in air samples. The assay detected fewer than 10 A. fumigatus conidia per qPCR reaction and quantified conidia over a 4-log10 range with high linearity (R2 >0.99) and low variability among replicate standards (CV=2.0%) in less than 4 h. The sensitivity and linearity of qPCR for conidia deposited on filters was equivalent to conidia calibration standards. A. fumigatus DNA from 8 isolates was consistently quantified using this method, while non-specific DNA from 14 common environmental fungi, including 6 other Aspergillus species, was not detected. This method provides a means of analyzing long term air samples collected on filters which may enable investigators to correlate airborne environmental A. fumigatus conidia concentrations with adverse health effects.
Air↗
Laboratory efforts to cultivate noroviruses.
Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide and are recognized as the foremost cause of foodborne illness. Despite numerous efforts, routine cell cultures have failed to yield replicating NoV. This paper describes methods used to try to grow NoV in vitro in two laboratories. Cells (A549, AGS, Caco-2, CCD-18, CRFK, CR-PEC, Detroit 551, Detroit 562, FRhK-4, HCT-8, HeLa, HEC, HEp-2, Ht-29, HuTu-80, I-407, IEC-6, IEC-18, Kato-3, L20B, MA104, MDBK, MDCK, RD, TMK, Vero and 293) were cultured on solid or permeable surfaces. Differentiation was induced using cell culture supplements such as insulin, DMSO and butyric acid. In some cases, the cells and the NoV-containing stool samples were treated with bioactive digestive additives. Variables evaluated in cultivation experiments included the method of preparation of the virus inoculum, the genotype of the virus, conditions for maintenance of cell monolayers, additives in the maintenance medium and the method of inoculation of the cells. Serial blind passage studies were performed routinely. In addition to evaluation for CPE, evidence of virus replication was sought using immunofluorescent assays to detect newly produced viral capsid antigen and RT-PCR assays to detect the viral genome. Although some infected cultures remained NoV positive by RT-PCR for up to five passages and an occasional cell in a monolayer showed evidence of specific immunofluorescence, no reproducible NoV-induced CPE was observed and all RT-PCR results that were positive initially were negative following continued passaging. Thus, attempts to develop a method for the cultivation of NoV were unsuccessful.
Animals↗
Development of a PCR-enzyme immunoassay oligoprobe detection method for Toxoplasma gondii oocysts, incorporating PCR controls.
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, uracil-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.
Animals↗