scholarly journals Quantification of Enteric Viruses, Pathogen Indicators, and Salmonella Bacteria in Class B Anaerobically Digested Biosolids by Culture and Molecular Methods

2010 ◽  
Vol 76 (19) ◽  
pp. 6441-6448 ◽  
Author(s):  
Kelvin Wong ◽  
Brandon M. Onan ◽  
Irene Xagoraraki
Keyword(s):  
Salmonella Enterica ◽  
Hepatitis A Virus ◽  
A549 Cells ◽  
Enteric Viruses ◽  
The United States ◽  
Land Application ◽  
Most Probable Number ◽  
Probable Number ◽  
Pathogen Indicators ◽  
Class B

ABSTRACT The most common class B biosolids in the United States are generated by mesophilic anaerobic digestion (MAD), and MAD biosolids have been used for land application. However, the pathogen levels in MAD biosolids are still unclear, especially with respect to enteric viruses. In this study, we determined the occurrence and the quantitative levels of enteric viruses and indicators in 12 MAD biosolid samples and of Salmonella enterica in 6 MAD biosolid samples. Three dewatered biosolid samples were also included in this study for purposes of comparison. Human adenoviruses (HAdV) had the highest gene levels and were detected more frequently than other enteric viruses. The gene levels of noroviruses (NV) reported were comparable to those of enteroviruses (EV) and human polyomaviruses (HPyV). The occurrence percentages of HAdV, HAdV species F, EV, NV GI, NV GII, and HPyV in MAD samples were 83, 83, 42, 50, 75, and 58%, respectively. No hepatitis A virus was detected. Infectious HAdV was detected more frequently than infectious EV, and all infectious HAdV were detected when samples were propagated in A549 cells. Based on most-probable-number (MPN) analysis, A549 cells were more susceptible to biosolid-associated viruses than BGM cells. All indicator levels in MAD biosolids were approximately 104 MPN or PFU per gram (dry), and the dewatered biosolids had significantly higher indicator levels than the MAD biosolids. Only two MAD samples tested positive for Salmonella enterica, where the concentration was below 1.0 MPN/4 g. This study provides a broad comparison of the prevalence of different enteric viruses in MAD biosolids and reports the first detection of noroviruses in class B biosolids. The observed high quantitative and infectivity levels of adenoviruses in MAD biosolids indicate that adenovirus is a good indicator for the evaluation of sludge treatment efficiency.

scholarly journals Detection of both Hepatitis A Virus and Norwalk-Like Virus in Imported Clams Associated with Food-Borne Illness

2002 ◽  
Vol 68 (8) ◽  
pp. 3914-3918 ◽  
Author(s):  
David H. Kingsley ◽  
Gloria K. Meade ◽  
Gary P. Richards
Keyword(s):  
Hepatitis A ◽  
Magnetic Beads ◽  
Hepatitis A Virus ◽  
New York State ◽  
Rna Extraction ◽  
The United States ◽  
Fecal Coliforms ◽  
Most Probable Number ◽  
Genotype I ◽  
Probable Number

ABSTRACT Hepatitis A virus (HAV) and Norwalk-like virus (NLV) were detected by reverse transcription-PCR in clams imported into the United States from China. An epidemiological investigation showed that these clams were associated with five cases of Norwalk-like gastroenteritis in New York State in August 2000 (Food and Drug Administration Import Alert 16-50). They were labeled “cooked” but appeared raw. Viral RNA extraction was performed by using dissected digestive tissues rather than whole shellfish meats; this was followed by glycine buffer elution, polyethylene glycol precipitation, Tri-Reagent treatment, and purification of poly(A) RNA with magnetic beads coupled to poly(dT) oligonucleotides. We identified HAV and NLV as genotype I and genogroup II strains, respectively. Both viruses have high levels of homology to Asian strains. An analysis of fecal coliforms revealed a most-probable number of 93,000/100 g of clam meat, which is approximately 300-fold higher than the hygienic standard for shellfish meats.

scholarly journals Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin

2003 ◽  
Vol 69 (2) ◽  
pp. 1172-1180 ◽  
Author(s):  
Mark A. Borchardt ◽  
Phil D. Bertz ◽  
Susan K. Spencer ◽  
David A. Battigelli
Keyword(s):  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
The United States ◽  
Land Application ◽  
Private Household ◽  
Septic Systems ◽  
Rt Pcr ◽  
Sequential Samples ◽  
Human Enteric Viruses ◽  
Public Water Systems

ABSTRACT Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells. The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems. Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season. Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs). In addition, cell culture was used to detect culturable enteroviruses. Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses. Among the 50 wells, four (8%) were positive for viruses by RT-PCR. Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample. Contamination was transient, since none of the wells was virus positive for two sequential samples. Culturable enteroviruses were not detected in any of the wells. Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride. The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses.

scholarly journals Salmonella enterica Burden in Harvest-Ready Cattle Populations from the Southern High Plains of the United States

2007 ◽  
Vol 74 (2) ◽  
pp. 345-351 ◽  
Author(s):  
David J. Kunze ◽  
Guy H. Loneragan ◽  
Tammy M. Platt ◽  
Mark F. Miller ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
United States ◽  
Salmonella Enterica ◽  
The United States ◽  
Most Probable Number ◽  
High Plains ◽  
Antimicrobial Drugs ◽  
Probable Number ◽  
At Risk Populations ◽  
High Level ◽  
Level Resistance

ABSTRACT Our objectives were to quantify the Salmonella enterica burdens in harvest-ready cattle and to identify specific at-risk populations of cattle most likely to harbor multiply resistant S. enterica. Hide swabs were collected in abattoirs from three cohorts of cattle (feedlot origin cattle that had achieved desirable harvest characteristics and dairy- and beef-type cows harvested because of poor productivity). Feces were collected from two cohorts housed in feedlots (cattle that had achieved desirable harvest characteristics and animals identified for salvage recovery because of poor productivity). Facilities were visited on four occasions over a 12-month period. Salmonella enterica isolates were recovered, and organisms were quantified using standard microbiological methodologies. Susceptibility to antimicrobial drugs and serotype were determined for one S. enterica isolate per sample. Salmonella enterica was recovered from 55.6% of 1,681 samples. The prevalences on hides and in feces were 69.6% and 30.3%, respectively. The concentrations of S. enterica organisms averaged (as determined by the most probable number technique) 1.82 log10/100 cm2 of hides and 0.75 log10/g of feces. None of the isolates recovered from cattle that had achieved desirable harvest characteristics were resistant to four or more drugs. For isolates recovered from animals with poor productivity characteristics, 6.5% were resistant to four or more drugs. Twenty-two serovars were identified, with the most common being Salmonella enterica serovar Anatum (25.5%), Salmonella enterica serovar Montevideo (22.2%), and Salmonella enterica serovar Cerro (12.5%). High-level resistance, i.e., resistance to four or more drugs, was clustered within a few relatively uncommon serovars. These results demonstrate that even though S. enterica isolates are readily recoverable from harvest-ready cattle, multiply resistant variants are rare and are associated with specific serovars in cattle harvested because of poor productivity characteristics.

A risk assessment of emerging pathogens of concern in the land application of biosolids

2002 ◽  
Vol 46 (10) ◽  
pp. 225-230 ◽  
Author(s):  
C.P. Gerba ◽  
I.L. Pepper ◽  
L.F. Whitehead
Keyword(s):  
Risk Model ◽  
Hepatitis A Virus ◽  
The United States ◽  
Land Application ◽  
Pathogen Transmission ◽  
Emerging Pathogens ◽  
Response Models ◽  
Treatment Processes ◽  
Class B ◽  
Moisture Conditions

Since the development of the United States Environmental Protection Agency's 503 biosolids Rule, which includes treatment requirements to reduce the threat of pathogen transmission, many new pathogens have been recognized which could be transmitted by biosolids. A risk analysis was performed to assess which emerging pathogens would be most likely to survive treatments required for Class B biosolids before land application. The literature was reviewed on the resistance of emerging pathogens to temperature and other environmental factors to assess their probability of surviving various biosolids treatment processes. In addition existing information on occurrence in biosolids and dose response models for each pathogen was reviewed. It was concluded that adenoviruses and hepatitis A virus are the most thermally resistant viruses and can survive for prolonged periods in the environment. The protozoan parasites microsporidia and Cyclospora were unlikely to survive the temperatures achieved in anaerobic digestion and do not survive well under low moisture conditions. A risk model was used to assess the risk of infection and illness from enteric viruses after application of class B biosolids.

Persistence of Orally Administered Salmonella enterica Serovars Agona and Montevideo in Atlantic Salmon (Salmo salar L.)

2005 ◽  
Vol 68 (7) ◽  
pp. 1336-1339 ◽  
Author(s):  
L. L. NESSE ◽  
T. LØVOLD ◽  
B. BERGSJØ ◽  
K. NORDBY ◽  
C. WALLACE ◽  
...  
Keyword(s):  
Gastrointestinal Tract ◽  
Atlantic Salmon ◽  
Salmo Salar ◽  
Salmonella Enterica ◽  
Most Probable Number ◽  
Fish Feed ◽  
Internal Organs ◽  
Probable Number ◽  
Atlantic Salmon Salmo Salar ◽  
Gastric Intubation

The objective of our experiments was to study the persistence and dissemination of orally administered Salmonella in smoltified Atlantic salmon. In experiment 1, salmon kept at 15°C were fed for 1 week with feed contaminated with 96 most-probable-number units of Salmonella Agona per 100 g of feed and then starved for 2 weeks. Samples were taken from the gastrointestinal tract and examined for Salmonella 1, 2, 8, 9, 15, and 16 days after the feeding ended. In experiment 2, Salmonella Agona and Montevideo were separately mixed with feed and administered by gastric intubation. Each fish received 1.0 × 108, 1.0 × 106, or 1.0 × 104 CFU. The different groups were kept in parallel at 5 and 15°C and observed for 4 weeks. Every week, three fish in each group were sacrificed, and samples were taken from the skin, the pooled internal organs, the muscle, and the gastrointestinal tract and examined for the presence of Salmonella. The results from the two experiments showed that the persistence of Salmonella in the fish was highly dependent on the dose administered. Salmonella was not recovered from any of the fish that were fed for 1 week with the lowest concentration of Salmonella. In the fish given the highest dose of Salmonella, bacteria persisted for at least 4 weeks in the gastrointestinal tract as well as, to some extent, the internal organs. The present study shows that under practical conditions in Norway, the risk of Salmonella in fish feed being passed on to the consumer of the fish is negligible.

Fecal Coliform Methods for Examination of Sea Water: Interlaboratory Evaluation of Split Sample Analysis

1978 ◽  
Vol 61 (4) ◽  
pp. 772-778
Author(s):  
John J Miescier ◽  
Virgil E Carr ◽  
John F Musselman ◽  
Santo A Furfari
Keyword(s):  
Elevated Temperature ◽  
Sea Water ◽  
The United States ◽  
Most Probable Number ◽  
Test Procedures ◽  
Probable Number ◽  
E Coli ◽  
Split Sample ◽  
Elevated Temperature Test ◽  
Temperature Test

Abstract An interlaboratory study was conducted to compare the effectiveness of the following 3 multiple-tube fermentation methods for determining the most probable number (MPN) of Escherichia coli in a split artificial sea water sample: ( 1 ) the 72-hr standard methods procedure of the American Public Health Association, (2) a 24-hr elevated-temperature test using A-l medium, and (3) a 24-hr elevated temperature test modified to include an initial 3-hr resuscitation period using A-l medium. The capability of the laboratories to perform the 3 test procedures was also compared. Split sample replicates with low, medium, and high levels of E. coli were examined in 18 laboratories in the United States and Canada. Data indicate that the laboratories performed each test with equal capability, and all 3 procedures were equally effective in enumerating the strain of E. coli used in this investigation. By virtue of its homogeneity and stability, the split sample served as an appropriate specimen for this study and could probably be used as a proficiency test specimen for evaluating laboratory analyst performance in the bacteriological examination of sea water.

Survey of Postharvest-Processed Oysters in the United States for Levels of Vibrio vulnificus and Vibrio parahaemolyticus

2009 ◽  
Vol 72 (10) ◽  
pp. 2110-2113 ◽  
Author(s):  
ANGELO DePAOLA ◽  
JESSICA L. JONES ◽  
KATHY E. NOE ◽  
ROBIN H. BYARS ◽  
JOHN C. BOWERS
Keyword(s):  
Vibrio Vulnificus ◽  
Gulf Coast ◽  
Virulence Gene ◽  
Selective Advantage ◽  
The United States ◽  
Most Probable Number ◽  
Probable Number ◽  
Mild Heat ◽  
Thermostable Direct Hemolysin ◽  
The Mean

From June through October 2004, the U.S. Food and Drug Administration collected oysters (61 samples) that had been subjected to postharvest processing (PHP) methods, including mild heat treatment, freezing, and high hydrostatic pressure, from processors and retail markets in various states to determine Vibrio vulnificus and V. parahaemolyticus levels. Presence in a 25-g sample and most probable number (MPN) using standard enrichment and selective isolation procedures were utilized. Suspect colonies were isolated and identified using DNA probe colony hybridization. Neither species of vibrio was detected in 25-g portions of most samples regardless of the PHP. The lowest frequency of isolation of either pathogen (<10%) was observed with the mild heat process. Few (12 to 13%) frozen samples collected at the processor but not at retail contained >30 MPN/g of either pathogen. The mean levels of either organism in PHP oysters observed in the present study were 5 to 6 log less than in unprocessed raw Gulf Coast oysters. Of the 70 V. vulnificus isolates examined, only 5 possessed the putative virulence marker, type B 16S rRNA. Neither the thermostable direct hemolysin (tdh) nor the tdh-related hemolysin (trh) virulence gene was detected in any of the 40 V. parahaemolyticus isolates examined in the present study. These data suggest that if there is any selective advantage to pathogenic strains of V. vulnificus and V. parahaemolyticus, these differences are minimal. These results indicate that all PHP treatments greatly reduce exposure of V. vulnificus and V. parahaemolyticus to raw-oyster consumers. Consequently, these PHP oysters pose a much lower risk of illness to consumers due to these pathogens.

scholarly journals Bacterial and Viral Pathogens in Live Oysters: 2007 United States Market Survey

2010 ◽  
Vol 76 (9) ◽  
pp. 2754-2768 ◽  
Author(s):  
Angelo DePaola ◽  
Jessica L. Jones ◽  
Jacquelina Woods ◽  
William Burkhardt ◽  
Kevin R. Calci ◽  
...  
Keyword(s):  
Hepatitis A Virus ◽  
Control Measures ◽  
Fecal Coliforms ◽  
Seasonal Effects ◽  
Most Probable Number ◽  
Indicator Organisms ◽  
Probable Number ◽  
Detection Frequency ◽  
The Pacific ◽  
Two Samples

ABSTRACT Two samples of market oysters, primarily from retail establishments, were collected twice each month in each of nine states during 2007. Samples were shipped refrigerated overnight to five U.S. Food and Drug Administration laboratories on a rotating basis and analyzed by most probable number (MPN) for total and pathogenic Vibrio parahaemolyticus and V. vulnificus numbers and for the presence of toxigenic V. cholerae, Salmonella spp., norovirus (NoV), and hepatitis A virus (HAV). Levels of indicator organisms, including fecal coliforms (MPN), Escherichia coli (MPN), male-specific bacteriophage, and aerobic plate counts, were also determined. V. parahaemolyticus and V. vulnificus levels were distributed seasonally and geographically by harvest region and were similar to levels observed in a previous study conducted in 1998-1999. Levels of pathogenic V. parahaemolyticus were typically several logs lower than total V. parahaemolyticus levels regardless of season or region. Pathogenic V. parahaemolyticus levels in the Gulf and Mid-Atlantic regions were about two logs greater than the levels observed in the Pacific and North Atlantic regions. Pathogens generally associated with fecal pollution were detected sporadically or not at all (toxigenic V. cholerae, 0%; Salmonella, 1.5%; NoV, 3.9%; HAV, 4.4%). While seasonal prevalences of NoV and HAV were generally greater in oysters harvested from December to March, the low detection frequency obscured any apparent seasonal effects. Overall, there was no relationship between the levels of indicator microorganisms and the presence of enteric viruses. These data provide a baseline that can be used to further validate risk assessment predictions, determine the effectiveness of new control measures, and compare the level of protection provided by the U.S. shellfish sanitation system to those in other countries.

scholarly journals A Multiplex Reverse Transcription-PCR Method for Detection of Human Enteric Viruses in Groundwater

2003 ◽  
Vol 69 (6) ◽  
pp. 3158-3164 ◽  
Author(s):  
G. Shay Fout ◽  
Beth C. Martinson ◽  
Michael W. N. Moyer ◽  
Daniel R. Dahling
Keyword(s):  
Reverse Transcription ◽  
Hepatitis A Virus ◽  
Disease Outbreaks ◽  
Enteric Viruses ◽  
The United States ◽  
Norwalk Virus ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Groundwater Samples ◽  
Human Enteric Viruses

ABSTRACT Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed. The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group.

scholarly journals Dynamics of Salmonella Shedding and Welfare of Hens in Free-Range Egg Production Systems

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Vaibhav C. Gole ◽  
Rebecca Woodhouse ◽  
Charles Caraguel ◽  
Talia Moyle ◽  
Jean-Loup Rault ◽  
...  
Keyword(s):  
Production System ◽  
Salmonella Enterica ◽  
Egg Production ◽  
Production Systems ◽  
Most Probable Number ◽  
Probable Number ◽  
Content Type ◽  
Free Range ◽  
Phage Types ◽  
Positive Correlations

ABSTRACT The current study investigated the effect of environmental stressors (i.e., weather changes) on Salmonella shedding in free-range production systems and the correlations with behavioral and physiological measures (i.e., fecal glucocorticoid metabolites). This involved longitudinal and point-in-time surveys of Salmonella shedding and environmental contamination on four commercial free-range layer farms. The shedding of Salmonella was variable across free-range farms and in different seasons. There was no significant effect of season on the Salmonella prevalence during this investigation. In this study, the combined Salmonella most probable number (MPN) counts in environmental (including feces, egg belt, dust, nest box, and ramp) samples were highest in samples collected during the summer season (4th sampling, performed in February). The predominant serovars isolated during this study were Salmonella enterica serovar Mbandaka and Salmonella enterica serovar Typhimurium phage types 135 and 135a. These two phage types were involved in several egg product-related Salmonella outbreaks in humans. Multilocus variable-number tandem-repeat analysis (MLVA) results indicated that MLVA types detected from human food poisoning cases exhibited MLVA patterns similar to the strains isolated during this study. All Salmonella isolates (n = 209) were tested for 15 different genes involved in adhesion, invasion, and survival of Salmonella spp. We also observed variations for sopA, ironA, and misL. There were no positive correlations between fecal corticosterone metabolite (FCM) and Salmonella prevalence and/or shedding in feces. Also, there were no positive correlations between Salmonella prevalence and Salmonella count (log MPN) and any of the other welfare parameters. IMPORTANCE In this study, the welfare of laying hens and Salmonella shedding were compared over a prolonged period of time in field conditions. This study investigated the long-term shedding of Salmonella serovars in a free-range egg production system. Given that there is increasing demand for free-range eggs, it is essential to understand the risks associated with such a production system.

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