New USEPA water quality criteria by 2012: GOMA concerns and recommendations.

The Gulf of Mexico Alliance (GOMA) was tasked by the five Gulf State Governors to identify major issues affecting the Gulf of Mexico (GoM) and to set priorities for ameliorating these problems. One priority identified by GOMA is the need to improve detection methods for water quality indicators, pathogens and microbial source tracking. The United States Environmental Protection Agency (USEPA) is tasked with revising water quality criteria by 2012; however, the locations traditionally studied by the USEPA are not representative of the GoM and this has raised concern about whether or not the new criteria will be appropriate. This paper outlines a number of concerns, including deadlines associated with the USEPA Consent Decree, which may prevent inclusion of research needed to produce a well-developed set of methods and criteria appropriate for all regulated waters. GOMA makes several recommendations including ensuring that criteria formulation use data that include GoM-specific conditions (e.g. lower bather density, nonpoint sources), that rapid-testing methods be feasible and adequately controlled, and that USEPA maintains investments in water quality research once the new criteria are promulgated in order to assure that outstanding scientific questions are addressed and that scientifically defensible criteria are achieved for the GoM and other regulated waterbodies.

Salmonella rarely detected in Mississippi coastal waters and sediment.

AIMS: Standards for the rapid detection of individual pathogens from environmental samples have not been developed, but in their absence, the use of molecular-based detection methods coupled with traditional microbiology techniques allows for rapid and accurate pathogen detection from environmental waters and sediment. The aim of this research was to combine the use of enrichment with PCR for detection of Salmonella in Mississippi coastal waters and sediment and observe if that presence correlated with levels of enterococci and climatological variables. METHODS AND RESULTS: Salmonella were primarily found in samples that underwent nutrient enrichment and were present more frequently in freshwater than marine waters. Salmonella were detected infrequently in marine and freshwater sediments. There was a significant positive correlation between the presence of detectable Salmonella and the average enterococcal count. An inverse relationship, however, was observed between the frequency of detection and the levels of salinity, turbidity and sunlight exposure. CONCLUSIONS: Results from this study indicated the presence of Salmonella in Mississippi coastal waters, and sediments are very low with significant differences between freshwater and marine environments. SIGNIFICANCE AND IMPACT OF THE STUDY: Using pathogenic and novel nonpathogenic molecular markers, Salmonella do not appear to be a significant pathogenic genus along the Mississippi Coast.

Validation and field testing of library-independent microbial source tracking methods in the Gulf of Mexico.

Water quality is frequently impacted by microbial pollution from human and animal feces. Microbial source tracking (MST) can identify dominant pollution sources and improve assessment of health risk compared to indicator bacteria alone. This study aims to standardize and validate MST methods across laboratories in coastal Gulf of Mexico states. Three laboratories evaluated library-independent MST methods for human sewage detection via conventional PCR: (1) human-associated Bacteroidales, (2) human polyomaviruses (HPyVs), and (3) Methanobrevibacter smithii. All methods detected targets in human sewage seeded into buffer, freshwater or marine water (100% sensitivity). The limit of detection (LOD) for human sewage was lowest for the Bacteroidales assay (10(-5)-10(-6) dilution). LODs for HPyVs and M. smithii assays were similar to each other (10(-3)-10(-4)), but were higher than Bacteroidales. The HPyVs assay was 100% specific, showing no cross-reactivity to dog, cow, cat, bird, or wild animal feces among >300 samples from three Gulf Coast regions. The human Bacteroidales assay was 96% specific, but cross-reacted with 10% of dog and some chicken samples. The M. smithii assay was 98% specific with limited cross-reactivity with cow, dog and seagull samples. An experts' workshop concluded that all methods showed sufficient accuracy and reliability to move forward. SOPs will be distributed to collaborating laboratories for further inter-laboratory comparison, and field validation will occur in year 2.

A statistical appraisal of disproportional versus proportional microbial source tracking libraries.

Library-based microbial source tracking (MST) can assist in reducing or eliminating fecal pollution in waters by predicting sources of fecal-associated bacteria. Library-based MST relies on an assembly of genetic or phenotypic "fingerprints" from pollution-indicative bacteria cultivated from known sources to compare with and identify fingerprints of unknown origin. The success of the library-based approach depends on how well each source candidate is represented in the library and which statistical algorithm or matching criterion is used to match unknowns. Because known source libraries are often built based on convenience or cost, some library sources may contain more representation than others. Depending on the statistical algorithm or matching criteria, predictions may become severely biased toward classifying unknowns into the library's dominant source category. We examined prediction bias for four of the most commonly used statistical matching algorithms in library-based MST when applied to disproportionately-represented known source libraries; maximum similarity (MS), average similarity (AS), discriminant analyses (DA), and k-means nearest neighbor (k-NN). MS was particularly sensitive to disproportionate source representation. AS and DA were more robust. k-NN provided a compromise between correct prediction and sensitivity to disproportional libraries including increased matching success and stability that should be considered when matching to disproportionally-represented libraries.

Methanobrevibacter ruminantium as an indicator of domesticated-ruminant fecal pollution in surface waters.

A PCR-based assay (Mrnif) targeting the nifH gene of Methanobrevibacter ruminantium was developed to detect fecal pollution from domesticated ruminants in environmental water samples. The assay produced the expected amplification product only when the reaction mixture contained DNA extracted from M. ruminantium culture, bovine (80%), sheep (100%), and goat (75%) feces, and water samples from a bovine waste lagoon (100%) and a creek contaminated with bovine lagoon waste (100%). The assay appears to be specific and sensitive and can distinguish between domesticated- and nondomesticated-ruminant fecal pollution in environmental samples.

Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes.

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.

Fidelity of bacterial source tracking: Escherichia coli vs Enterococcus spp and minimizing assignment of isolates from nonlibrary sources.

AIMS: The goal of the study was to improve the fidelity of library-dependent bacterial source tracking efforts in determining sources of faecal pollution. The first objective was to compare the fidelity of source assignments using Escherichia coli vs Enterococcus spp. The second objective was to determine the efficacy of using thresholds during source assignments to reduce the rate of misassignments when nonlibrary isolates (i.e. isolates from animals not used in building the identification library) are present. METHODS AND RESULTS: E. coli and Enterococcus isolates from 784 human, cow, deer, dog, chicken, and gull faecal samples were fingerprinted using BOX-PCR. Jack-knife analysis of the fingerprints showed that the overall rate of correct assignment (ORCA) of 867 E. coli isolates was 67% compared with 82% for 1020 Enterococcus isolates. In a separate blind test using similarity value and quality factor thresholds, the ORCA of 130 E. coli and 131 Enterococcus isolates were 70% and 98%, respectively. The use of these thresholds reduced misassignment of 262 nonlibrary enterococcal isolates from horses, goats, pigs, bats, squirrels, ducks, geese, and migratory song birds. Misassignment was reduced from 100% when thresholds were not used, to 47% using similarity threshold alone, and to 12% when both thresholds were used. CONCLUSIONS: The use of enterococci provides higher rates of correct source assignment compared with E. coli. The use of similarity thresholds to decide whether to accept source assignments made by computer programmes reduces the rate of misassignment of nonlibrary isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Although both E. coli and Enterococcus spp. are still used in microbial source tracking, the use of enterococci should be preferred over the use of E. coli in DNA fingerprint-based efforts. In addition, because environmental isolates are not limited to those from animals used to build source tracking libraries, similarity thresholds should be used during source assignments to reduce the rate of misassignments.

Detection of the nifH gene of Methanobrevibacter smithii: a potential tool to identify sewage pollution in recreational waters.

AIMS: The goal of this study was to develop and test the efficacy of a PCR assay for the environmental detection of the nifH gene of Methanobrevibacter smithii, a methanogen found in human faeces and sewage. METHODS AND RESULTS: PCR primers for the nifH gene of M. smithii were designed, tested and used to detect the presence or absence of this organism in faecal and environmental samples. Specificity analysis showed that the Mnif primers amplified products only in M. smithii pure culture strains (100%), human faeces (29%), human sewage samples (93%) and sewage-contaminated water samples (100%). No amplification was observed when primers were tested against 43 bacterial stock cultures, 204 animal faecal samples, 548 environmental bacterial isolates and water samples from a bovine waste lagoon and adjacent polluted creek. Sequencing of PCR products from sewers demonstrated that a 222-bp product was the nifH gene of M. smithii. The minimal amount of total DNA required for the detection of M. smithii was 10 ng for human faeces, 10 ng for faecally contaminated water and 5 ng for sewage. Recreational water seeded with M. smithii established a lower detection limit of 13 cells ml(-1). CONCLUSIONS: The Mnif assay developed during this investigation showed successful detection of M. smithii in individual human faecal samples, sewage and sewage-contaminated water but not in uncontaminated marine water or bovine-contaminated waters. The Mnif assay appears to be a potentially useful method to detect sewage-polluted coastal waters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to utilize methanogens as an indicator of sewage pollution. Mnif PCR detection of M. smithii was shown to be a rapid, inexpensive and reliable test for determining the presence or absence of sewage pollution in coastal recreational waters.

Phenotypic library-based microbial source tracking methods: efficacy in the California collaborative study.

As part of a larger microbial source tracking (MST) study, several laboratories used library-based, phenotypic subtyping techniques to analyse fecal samples from known sources (human, sewage, cattle, dogs and gulls) and blinded water samples that were contaminated with the fecal sources. The methods used included antibiotic resistance analysis (ARA) of fecal streptococci, enterococci, fecal coliforms and E. coli; multiple antibiotic resistance (MAR) and Kirby-Bauer antibiotic susceptibility testing of E. coli; and carbon source utilization for fecal streptococci and E. coli. Libraries comprising phenotypic patterns of indicator bacteria isolated from known fecal sources were used to predict the sources of isolates from water samples that had been seeded with fecal material from the same sources as those used to create the libraries. The accuracy of fecal source identification in the water samples was assessed both with and without a cut-off termed the minimum detectable percentage (MDP). The libraries (approximately 300 isolates) were not large enough to avoid the artefact of source-independent grouping, but some important conclusions could still be drawn. Use of a MDP decreased the percentage of false-positive source identifications, and had little effect on the high percentage of true-positives in the most accurate libraries. In general, the methods were more prone to false-positive than to false-negative errors. The most accurate method, with a true-positive rate of 100% and a false-positive rate of 39% when analysed with a MDP, was ARA of fecal streptococci. The internal accuracy of the libraries did not correlate with the accuracy of source prediction in water samples, showing that one should not rely solely on parameters such as the average rate of correct classification of a library to indicate its predictive capabilities.

Assessment of statistical methods used in library-based approaches to microbial source tracking.

Several commonly used statistical methods for fingerprint identification in microbial source tracking (MST) were examined to assess the effectiveness of pattern-matching algorithms to correctly identify sources. Although numerous statistical methods have been employed for source identification, no widespread consensus exists as to which is most appropriate. A large-scale comparison of several MST methods, using identical fecal sources, presented a unique opportunity to assess the utility of several popular statistical methods. These included discriminant analysis, nearest neighbour analysis, maximum similarity and average similarity, along with several measures of distance or similarity. Threshold criteria for excluding uncertain or poorly matched isolates from final analysis were also examined for their ability to reduce false positives and increase prediction success. Six independent libraries used in the study were constructed from indicator bacteria isolated from fecal materials of humans, seagulls, cows and dogs. Three of these libraries were constructed using the rep-PCR technique and three relied on antibiotic resistance analysis (ARA). Five of the libraries were constructed using Escherichia coli and one using Enterococcus spp. (ARA). Overall, the outcome of this study suggests a high degree of variability across statistical methods. Despite large differences in correct classification rates among the statistical methods, no single statistical approach emerged as superior. Thresholds failed to consistently increase rates of correct classification and improvement was often associated with substantial effective sample size reduction. Recommendations are provided to aid in selecting appropriate analyses for these types of data.

Recommendations for microbial source tracking: lessons from a methods comparison study.

The methods comparison study described in accompanying manuscripts demonstrated the potential value of microbial source tracking (MST) techniques, but also identified a need for method refinement. This paper provides three classes of recommendations to improve MST technology: optimization, development and evaluation. Optimization recommendations focus on library-dependent methods and include improved selection of restriction enzymes or antibiotics, better definition of appropriate library size, selection of target species and choice of statistical pattern-matching algorithms. Methods development recommendations focus on identifying new genomic targets and quantification procedures for library-independent methods. Longer-term methods development recommendations include integration of microarrays and other direct pathogen detection technology with MST. Studies defining host specificity and population dynamics should aid selection of target species during methods development. Evaluation recommendations include enhancements that should be incorporated into future methods comparison studies, along with studies to assess the value of MST results for risk characterization.

Cell cycle components and their potential impact on the development of continuous in vitro penaeid cell replication.

In vitro prawn cell culture has yet to produce an established cell line. In an effort to establish some understanding of the cellular blockage that prohibits their division in vitro we conducted several studies to characterize the cytoskeletal components of hemocytes and found no cells undergoing mitosis. Following this discovery, a molecular analysis of cell division regulatory proteins was performed. Cell cycle regulatory proteins (cyclins) have been identified as essential components in the progression of all eukaryotic cells through the cell cycle. We report here the identification of cyclin A and cyclin B proteins and their cofactor (p34(cdc2)) in making up the mitosis promoting factor (MPF) in protein extracts from egg and muscle tissues of Penaeus vannamei. Molecular weight analysis confirmed the size of the target proteins to be similar to the same proteins identified in the Atlantic surf clam (Spisula solidissima).

Relaying to Decrease the Concentration of Oyster-Associated Pathogens.

Oysters experimentally contaminated with indicator bacteria, Salmonella and poliovirus were used in relaying studies designed to measure microbial elimination under a variety of environmental conditions. Two factors, level of microorganism in the oyster and temperature of the water, were important in determining the length of time necessary to purge the contaminating organisms. Oysters under physiological stress cleansed at a slower rate than did healthy oysters. Based on the expected level of pathogen contamination in naturally polluted oysters, healthy relaid oysters were capable of cleansing in a 7-d period provided the temperature was above 10°C. These results were verified by following the elimination of indicator bacteria and poliovirus in commercially relaid oysters. Fecal indicator bacteria and enteric pathogenic bacteria were eliminated at similar rates but fecal coliform levels did not correlate with virus elimination. Relaying waters may contain some indicator bacteria and this study suggested that fecal coliforms may not be useful as end-point indicators for this method of oyster purification.

Elution of enteric viruses from Mississippi estuarine sediments with lecithin-supplemented eluents.

Isoelectric casein supplemented with lecithin was tested for its ability to recover enteric viruses from estuarine sediments of varied sand, silt, and clay composition. Recoveries were higher when lecithin was incorporated into an eluent as compared with trials with only the casein solution. Semipurified soybean lecithin (3%) allowed the highest overall recovery of virus from all sediments tested; crude soybean lecithin produced the lowest recovery. A difference in the percentage of virus able to be recovered from a sediment was related to the percentage of clay in the sample. Correlational statistics indicated a trend toward lower virus recovery as the clay composition of a sediment increased. Virus adsorption to the four sediments tested revealed differences between poliovirus, coxsackievirus, and echovirus adsorption that could not be explained on the basis of the clay content of a sediment.

Primary cell cultures from the teleost, Cyprinodon variegatus: culture establishment and application in carcinogen exposure studies.

Methods were developed for aseptic maintenance of Cyprinodon variegatus fry for extended periods. Preliminary studies indicated that under optimum conditions sterile embryos develop normally for a sufficient time to function as carcinogen-teratogen assay systems. An embryo-primary cell culture technique was developed that incorporates, in a single system, certain characteristics of both intact embryos and primary cell cultures and allows simultaneous observation of the effects of carcinogens on the whole organism and primary cell monolayers. The effective use of these systems provides one the opportunity to study the effects of carcinogens on teleosts at the cellular and organismic level.

Elution of viruses from coastal sediments.

Enteric viruses were eluted from estuarine sediments by using four organic mixtures; these solutions, with or without various supplements, were compared by determining their abilities to desorb virus from sediments taken from shellfish-harvesting sites. The least effective eluents consisted of glycine buffer, milk preparations, and beef extract paste. When virus type and sediment composition were taken into consideration, higher percentages of virus recovery were achieved with isoelectric casein, powdered beef extract, and nutrient broth mixtures. In addition to the type of eluent used, variations in virus recovery were due to the pH of the eluent, the composition of the sediment, and the type of virus being extracted. No clear distinction between the values of protein and inorganic ion supplements could be made.

Methods to Detect Viruses in Foods: Testing and Interpretation of Results 1.

Viruses that may be detected in foods should be considered pathogenic and treated with appropriate caution. In this discussion, specific procedures for extracting viruses from shellfish are presented for each of the major commercial species of bivalve molluscs. Other foods for which specific extraction methods are detailed include lettuce, frozen strawberries, ground beef and raw milk. Viruses that may be detected by the methods described are those which are capable of producing a perceptible effect while replicating in cultured primate cells. Both results that are apparently positive and those that are apparently negative require careful interpretation; one must be extremely skeptical if large numbers of food samples obtained at the market appear to yield viruses. The procedures that are now available have some important limitations, including inability to detect the viruses that cause most of the reported foodborne disease. Approaches to surmounting these limitations include use of serologic methods to detect viruses that do not cause perceptible effects in cell cultures and improvement of procedures for extracting all viruses from food samples.

Methods for Detecting Viruses in Foods: Background and General Principles 1.

Viruses are sometimes transmitted through foods. Hepatitis A (HA) and some viral gastroenteritides are the diseases now known to be spread most frequently in this way, but any virus from the human intestines probably could be. Bacterial indicators of fecal contamination show limited correlation with the incidence of viruses in foods, so tests to detect the viruses themselves are needed. The epidemiologic record has led to selection of shellfish (bivalve molluscs) and vegetables and fruits as foods for which test methods should be described, and ground beef and raw milk are also considered here because of the great interest they have attracted among research workers. Viruses in foods are presently detected on the basis of the infections they cause in cultured primate cells, but these cell culture methods do not permit detection of the HA virus nor the most important of the foodborne gastroenteritis viruses. Current methods of virus detection entail liquefaction of the food sample, clarification of the sample suspension, possibly concentration of the clarified food extract, and inoculation of cell cultures; a certain amount of specialized equipment is required for some of these procedures. The inclusion of the proper controls is critical to interpretation of results that are obtained.

Isolation of enteroviruses from oxidation pond waters.

Enteroviruses were isolated from influent and effluent wastewaters of two types of oxidation lagoons during an 18-month study. Isolations, performed by sewage concentration and direct assay, were low in number and did not follow a seasonal trend. The younger treatment system, using aeration and effluent chlorination, was more efficient at removing viruses than the aging, static complex.