F-Specific RNA Bacteriophages, Especially Members of Subgroup II, Should Be Reconsidered as Good Indicators of Viral Pollution of Oysters.

Norovirus (NoV) is the leading cause of gastroenteritis outbreaks linked to oyster consumption. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as indicators of viral contamination in oysters by focusing especially on FRNAPH subgroup II (FRNAPH-II). These viral indicators have been neglected because their behavior is sometimes different from that of NoV in shellfish, especially during the depuration processes usually performed before marketing. However, a significant bias needs to be taken into account. This bias is that, in the absence of routine culture methods, NoV is targeted by genome detection, while the presence of FRNAPH is usually investigated by isolation of infectious particles. In this study, by targeting both viruses using genome detection, a significant correlation between the presence of FRNAPH-II and that of NoV in shellfish collected from various European harvesting areas impacted by fecal pollution was observed. Moreover, during their depuration, while the long period of persistence of NoV was confirmed, a similar or even longer period of persistence of the FRNAPH-II genome, which was over 30 days, was observed. Such a striking genome persistence calls into question the relevance of molecular methods for assessing viral hazards. Targeting the same virus (i.e., FRNAPH-II) by culture and genome detection in specimens from harvesting areas as well as during depuration, we concluded that the presence of genomes in shellfish does not provide any information on the presence of the corresponding infectious particles. In view of these results, infectious FRNAPH detection should be reconsidered as a valuable indicator in oysters, and its potential for use in assessing viral hazard needs to be investigated.IMPORTANCE This work brings new data about the behavior of viruses in shellfish, as well as about the relevance of molecular methods for their detection and evaluation of the viral hazard. First, a strong correlation between the presence of F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and that of norovirus (NoV) in shellfish impacted by fecal contamination has been observed when both viruses are detected using molecular approaches. Second, when reverse transcription-PCR and culture are used to detect FRNAPH-II in shellfish, it appears that the genomes of the viruses present a longer period of persistence than infectious virus, and thus, virus genome detection fails to give information about the concomitant presence of infectious viruses. Finally, this study shows that FRNAPH persist at least as long as NoV does. These data are major arguments to reconsider the potential of FRNAPH as indicators of shellfish viral quality.

Seasonal gonadal development and age-related maturity patterns of introduced pumpkinseed (Lepomis gibbosus Linnaeus, 1758) in a heated thermal reservoir and an adjacent river reach.

Testis and ovarian maturation status, maturity profile and gonado-somatic index (GSI) were assessed in pumpkinseed (Lepomis gibbosus) collected from Mirgenbach, a cooling-water reservoir associated with a nuclear power plant, and from the River Moselle 7km downstream of the reservoir's thermal outflow. Histological investigation indicated that in both sexes, gonadal development of pumpkinseed in the heated reservoir was more advanced than in the cooler Moselle River throughout the breeding season. The histological maturity profile of reservoir males ranked by the advancement of sperm cells was highly correlated with its GSI (rs=0.73, P<0.001). GSI of females in the reservoir increased with the stage at maturity, but GSI was not significantly correlated with total length, age or growth rate of the individual. All sampled individuals of both sexes were mature at age 1 in the heated reservoir, whereas 48% of age 1 males and 57% of age 1 females were not mature in the river. GSI patterns suggest that males in the reservoir adopted one of two reproductive strategies (nesters or cuckolders), whereas no small males with large enough testes to be considered cuckolders were apparent in the river. The warm thermal regime of Mirgenbach Reservoir led to precocial maturity, early season reproduction, and the greater prevalence of apparent cuckolder males than would normally occur in this climatic zone.

Development of a viral concentration method for bottled water stored in hydrophobic support.

Several protocols have been described for the detection of genomes of enteric viruses from water using two-step procedures: membrane filtration and RT-PCR detection. However, these methods, when applied to bottled water, generally consider only the aqueous phase. Such procedures do not take into account the adhesion of viruses onto the hydrophobic container. Potential adhesion results in loss of viral concentration in the aqueous phase and consequently viral pollution is underestimated in such a system. A procedure based on the addition of surfactant to elute viruses followed by membrane concentration was developed to avoid this underestimation. Firstly, using poliovirus 1 as a model, this study demonstrated that the best solution to recover virus and/or viral genome is a mix of sodium dodecyl sulphate, a nonionic detergent and guanidine thiocyanate. Furthermore, temperature has a significant but low effect on elution. A positively charged 0.2 microm inorganic membrane composed of Alumina (Anodisc, Whatman) is also the best membrane to concentrate viral material before the detection by RT-PCR. Finally, the developed protocol gives significantly higher poliovirus 1 recovery rate than a reference protocol previously described (aqueous phase concentration on Zetapore). The difference can be explained by the recovery of the viruses adsorbed onto the water container.

An evolutionary interpretation of the significance of physical pain experienced by human females: defloration and childbirth pains.

The phenomena surrounding the pains commonly related to both first coitus and delivery have been addressed rather poorly in previous work, as regards their evolutionary aspects, during the investigation of human sexuality and reproductive behavior. In particular, the function of the hymen and the significance of defloration are largely misunderstood. The present paper aims to analyse the meaning of these two female physical pains in an evolutionary context. Accordingly, childbirth and defloration pains are hypothesized to manifest an adaptation designed to increase inclusive fitness in human evolutionary history. Clearly, the significance of pain as a message is essentially emotional. Indeed, the intense sexual emotions that may precede and/or follow the pain, the breaking and bleeding of the hymen during the first complete sexual act may generate distinctive strong feelings on/from each side of the newly formed couple. As to labor pain, both the shared intimacy with the mother and the emotional background during confinement may create mutual solicitude among the protagonists (i.e. midwifes, father, mother). Such feelings or attitudes may subsequently turn out to be beneficial to all of them, and more particularly to the newborn. As a general consequence, it appears that the two physical pains under consideration may have behavioral implications, in the sense that they contribute to increasing the stability of the connection between partners and thus, indirectly, to the welfare or even the survival (especially in former times) of the newborn child.

The glutathione-related detoxification pathway in the human breast: a highly coordinated system disrupted in the tumour tissues.

Glutathione and the associated enzymes, glutathione S-transferases, peroxidases, and reductase, have been implicated in cancer chemoresistance. This pathway was investigated in paired cancerous and peritumoral breast samples from 41 women. The tumours exhibited a higher redox status as deduced from increased transferase, peroxidase, and reductase activities and from higher total and reduced glutathione contents. Several components were strongly correlated in peritumoral tissues, suggesting a highly co-ordinated glutathione pathway that appeared disrupted in breast tumours with only a few correlations left. Therefore, resistance could spontaneously result from deregulated variations in the glutathione pathway, which might be relevant to the malignant disease progression.

The glutathione-related detoxification system is increased in human breast cancer in correlation with clinical and histopathological features.

PURPOSE: The glutathione detoxification pathway includes glutathione S-transferase (GST) and peroxidase (GPX) isoenzymes as well as glutathione reductase (GSSR). Though well established from cultured cancer cell lines, its involvement in resistance is still unclear in the tumours. This study aimed to describe the parameters that influence the glutathione contents and associated activities in breast cancer. METHODS: The components of the glutathione pathway were measured in the tumours from 41 women with primary breast cancer in comparison with those in the matched tumour-free samples. Appropriate statistical studies (regression analysis, Wilcoxon signed rank test) explored the influence of clinical and prognostic factors. RESULTS: Reduced and total glutathione contents were largely increased (P < 0.0001) and all related activities were significantly enhanced in the tumours. Interindividual variations were described, probably due to various parameters (age, menopause, axillary lymph node status, S and G2 + M cell fractions, ER, cathepsin-D and c-ErbB-2 expressions) that influence particular components of the glutathione pathway, especially the glutathione levels. CONCLUSIONS: The breast tumours improved their redox status and detoxification capacities depending on various parameters of significance for cell proliferation and aggressiveness, which supports the involvement of the glutathione pathway in malignant cell resistance to oxidative stress and apoptosis.

Comparative study of techniques used to recover viruses from residual urban sludge.

Eight virus extraction techniques were compared on three types of residual urban sludge for simultaneous detection of infectious enteroviruses, somatic coliphages, F-specific RNA bacteriophages and Bacteroides fragilis bacteriophages. The highest virus counts were found in extracts obtained using three extraction techniques described by respectively using a 10% beef extract solution at pH 9 and sonication, using a 0.3 M NaCl/7% beef extract solution at pH 7.5 and freon, and finally using a 0.1 M borate buffer/3% beef extract solution at pH 9 and sonication.

Enterovirus genomes in wastewater: concentration on glass wool and glass powder and detection by RT-PCR.

Standard methods for detecting enteroviruses in environmental samples require cell culture, which is time consuming and expensive. The reverse transcription-polymerase chain reaction (RT-PCR) is a rapid, sensitive method for detecting enteroviruses in water. However, environmental samples often contain substances that inhibit PCR amplification of target RNA. Hence the virus must be concentrated by procedures that do not interfere with amplification. This study shows that virus concentration by adsorption onto glass powder or glass wool supports is suitable for detecting viral genomes in treated wastewater by RT semi-nested PCR. No enterovirus genome was detected directly in 25 samples of treated wastewater by RT semi-nested PCR. However, samples concentrated by adsorption onto glass wool or glass powder showed that 48% (glass powder) and 56% (glass wool) contained virus. Secondary concentration by organic flocculation was unsuitable for detecting virus concentrated on glass wool (20% positive samples), but it helped to increase the detection of the genome after concentration on glass powder (72% positive samples).

Fates of bacteriophages and bacterial indicators in the Moselle river (France).

It has been suggested that bacteriophages can provide useful information about the pathogenic microorganisms, particularly enteric viruses, present in water. This information is complementary to that obtained from bacterial indicators of faecal contamination, which would be of great value for evaluating the risks associated with the use of certain types of water. Before bacteriophages can be used as indicators of faecal contamination, we need to confirm that bacteriophages give a different response to that given by the well-known bacteria indicators and to determine what happens to bacteriophages in river water. Indeed, drinking water is often produced from river water, either by natural filtration through the soil or after undergoing various treatments. We collected 96 river water samples from six different sites between February and November 2000. The samples were analysed for three faecal indicator bacteria (thermotolerant coliforms, enterococci and spores of sulphite-reducing anaerobes) and three types of bacteriophages (somatic coliphages, F-specific phages and Bacteroides fragilis phages). The densities of thermotolerant coliforms and enterococci depended mainly on physical factors such as flow rate and water temperature. High temperature and low flow rate led to a decrease in the density of these microorganisms, especially in the absence of a major input of faecal pollution. Conversely, the densities of somatic coliphages, F-specific phages and spores of sulphite-reducing anaerobes remained constant regardless of the flow rate and temperature. The density of Bacteroides fragilis phages was too low for unambiguous determination of their fate in river water.

Heterotrophic bacteria in water distribution systems. I. Spatial and temporal variation.

The drinking water distribution system of the city of Metz in France was sampled intensively during six, monthly surveys which were designed to determine the spatial and temporal distribution of total heterotrophic bacteria in the network. A non-hierarchical nearest-centroid clustering method was used for dividing the water distribution system into zones corresponding to different levels of bacterial density. The general pattern of the spatial heterogeneity showed a high degree of reproducibility. Since the frequency distribution of total heterotrophic bacteria within the zones was compatible with the negative binomial distribution, the water distribution system studied may be considered as being composed of several heterogeneous subsystems. The consistency of this structured spatial dispersion pattern of bacteria in light of some physical and chemical characteristics of the system is evident. In consideration of the principal features of flow in the system relevant to the layout of water mains, the location of zones of highest bacterial concentrations have been attributed to lower levels of chlorine residuals and prolonged retention time of the water in the network, especially in the storage units, before reaching the various distribution areas. Although the monthly variation in the bacterial concentration of the entire system showed a marked increase which was concomitant with warmest water temperatures, the zones were subject to noticeable discrepancies in the range of temporal variation.

Heterotrophic bacteria in water distribution systems. II. Sampling design for monitoring.

In this paper, which is a continuation of the work presented in Part I in this issue, previous information on the spatial and temporal variability of bacteriological data from a water distribution system is used to develop a sampling design for use in future water quality monitoring. The water distribution system is considered to be composed of several zones where the variation of bacterial counts in each zone is modelled by the negative binomial distribution. Under the assumption that the objective of monitoring is to determine whether or not the mean bacterial density of the water exceeds a specific standard, a criterion is given which determines the optimal number of sampling stations allocated to each zone. These stations are determined by assuming that either the risk of sampling (i.e. making the wrong decision) is prespecified or that the total number of stations to be sampled is predetermined. Sequential sampling to evaluate the compliance of the water with the standard is also discussed.

The use of historical data for estimating the number of samples required for monitoring drinking water.

Statistical techniques are described for estimating the number of samples required to monitor the quality of drinking water when the dispersion of bacteria in the water can be modeled by the Poisson or the negative binomial distributions. The concept of the operating characteristic (OC) curve of the water distribution system is presented and is used to evaluate the risk of declaring that the bacteriological water quality regulation is met when only a small portion of the water is analyzed. Assuming that the regulation requires that the monthly mean bacterial counts for samples of standard volume are to be less than one per ml, the OC curves are compared for different sample sizes and for different values of the parameters of the negative binomial. The results indicate that the correct specification of the model is very important in evaluating the risk of sampling (i.e. making the wrong decision). Total bacterial counts based on 1-ml samples, from the cities of Nancy and Metz in France, support the use of the negative binomial as a model for the dispersion of bacteria in drinking water. In the few cases where the negative binomial did not fit the data, the lack of fit can be attributed to the greater occurrence of the frequency of finding only one bacterium in the sample than that expected for the negative binomial. The OC curve indicated that the present monitoring strategy for the city of Nancy is adequate for monitoring the water quality if (i) the regulation requires that the monthly mean of total bacterial counts should not exceed one bacterium per ml, and (ii) the probability of accepting that the water quality is meeting the regulation, when the true mean number of bacteria per ml is two, should not be larger than 0.05. On the other hand, the city of Metz data indicated that it is necessary to increase the intensity of sampling both in time and space in order to achieve the same level of adequacy as that of the city of Nancy.

The effect of liming on the microbiological quality of urban sludge.

Treatments applied to sludge in order to stabilise and dehydrate them may give notable inactivation of microorganisms. This is observed when sludge is exposed either to high temperature or drastic pH when residual sludge is limed. The control of virological, parasitological and bacteriological sludge quality by detecting pathogenic microorganisms is slow and too expensive to be commonly practised. Thus, it is possible to replace pathogenic microorganisms detection by that of contamination indicators. The aim of this study was to determine the influence of liming on the behaviour of pathogenic microorganisms detected in urban sludge. The detection of Salmonella and helminth eggs was carried out in liquid sludge (2-3% dryness) and solid sludge (23% dryness) with added lime (0-45% weight/dry weight) and stored for 24 h-46 weeks. The results showed that liming modified some characteristics such as temperature, dryness and pH of the sludge. It appeared that, whatever the percentage of added lime, the temperature of liquid sludge did not change while it increased by about 9 degrees C when 30-45% lime was added to solid sludge. In the same way, the dryness of liquid sludge did not change during the liming, whereas the dryness of 45% limed solid sludge increased from 23% to 31%. Finally, 15%, 30% and 45% liming gave a pH of at least 10, 11.5 and 12, respectively, although the pH increase depended on the sludge type. The efficiency of liming was considered to be related to the pH and not to the percentage of added lime. Three factors determined the efficiency of pathogen elimination: (a) the pH reached by the sludge, (b) the period of liming activity and (c) the dryness of the sludge. Salmonella were eliminated from liquid sludge in 24 h at pH 10.7 and from solid sludge in 24 h at pH 10.0. Viable helminth egg concentration decreased to 3 eggs/10 g DM in liquid sludge in 14 d at pH 11.9 and 60 d at pH 11.6. In solid sludge, egg reduction was achieved in 24 h at pH 12.5, 7 d at pH 12.0 and 14 d at pH 11.5. From this study, it appeared that liming resulted in a much better microbiological quality of liquid sludge if its pH was maintained at 11.6 over 60 d or at pH 11.9 for 14 d. Solid sludge needed to be maintained at pH 11.5 for 14 d, pH 12.0 for 7 d or pH 12.5 for 24 h to achieve similar results.

Application of generalized linear models to the analysis of toxicity test data.

Generalized linear models give a unified approach to the performance of regression analysis of dichotomous, count or continuous data. This paper studies binomial, negative binomial and gamma regression models and gives a detailed description of inference procedures based on them. In particular, the maximum likelihood procedure is described for a logistic function (binomial regression) or a log-linear regression model (negative binomial and gamma regression). The process of model fitting and evaluation is illustrated by examples referring to the determination of endpoints in acute and chronic toxicity tests.

Analysis of two-way layout of count data with negative binomial variation.

A number of methods has been proposed for dealing with single-factor or factorial experiments when the requirements for performing the normal theory analysis of variance procedure are not satisfied. This paper suggests the use of the likelihood ratio statistic for testing the main effects and the interaction between the factors in two-way layout of count data following negative binomial distributions with a common dispersion parameter. The likelihood ratio statistic for testing the equality of the dispersion parameters of several groups of count data is also derived. The methods is illustrated by an example concerning the study of spatial and temporal variation of bacterial counts.

Comparison study on three protocols used to concentrate poliovirus type 1 from drinking water.

The efficiency of three techniques used to concentrate enteric viruses from water media and based on adsorption-elution on glass are tested. The techniques are adsorption on glass wool (GW) at the natural pH of the water and adsorption on glass powder using acidified water (pH 3.5). In the second case, two devices are used the classical apparatus (CGP) and the modified apparatus (MGP). A solution of glycine 0.05 M--3% beef extract pH 9.5 is used in all three techniques to perform the elution. The sensitivity of the above concentration methods is assayed with samples of 20 liters of tap water artificially contaminated with a known quantity of poliovirus type 1 (10(1) to 10(7) MPNCU [20 L]-1). The resulting concentrates are inoculated to BGM cell cultures and tittered according to the MPN technique. The study demonstrated that the recovery rate increased with the viral concentration of the samples with maximum efficiency reaching 81% for GW, 89% for CGP and 99% for MGP. A Wilcoxon test performed on paired samples and on the overall results with all three methods. Significant differences were demonstrated leading to the ranking of the techniques in the growing order of sensitivity GW, CGP and MGP. These finding were confirmed using a fitting technique according to the algorithm of Marquardt.

Rotavirus quantification: a comparison between the enumerative and the MPN techniques.

A commonly used technique to isolate rotaviruses from a water environment involves the inoculation on cell culture associated with the revelation of the viral multiplication by indirect immunofluorescence. The results of the experiment using this methodology may be expressed either with an enumerative technique or with a MPN technique. We have found that the enumerative method provided the highest yield as opposed to the MPN method by a ratio close to 3.7. According to the experimental conditions that were used, the enumerative method should be favored for the detection of viruses from a medium with low viral density or when quantifying viruses from a given sample. This work involves a comparative study where the MPN method is very attractive because of the ease of implementation and the rapidity with which results may be obtained.

Microplate fecal coliform method to monitor stream water pollution.

A study has been carried out on the Moselle River by means of a microtechnique based on the most-probable-number method for fecal coliform enumeration. This microtechnique, in which each serial dilution of a sample is inoculated into all 96 wells of a microplate, was compared with the standard membrane filter method. It showed a marked overestimation of about 14% due, probably, to the lack of absolute specificity of the method. The high precision of the microtechnique (13%, in terms of the coefficient of variation for log most probable number) and its relative independence from the influence of bacterial density allowed the use of analysis of variance to investigate the effects of spatial and temporal bacterial heterogeneity on the estimation of coliforms. Variability among replicate samples, subsamples, handling, and analytical errors were considered as the major sources of variation in bacterial titration. Variances associated with individual components of the sampling procedure were isolated, and optimal replications of each step were determined. Temporal variation was shown to be more influential than the other three components (most probable number, subsample, sample to sample), which were approximately equal in effect. However, the incidence of sample-to-sample variability (16%, in terms of the coefficient of variation for log most probable number) caused by spatial heterogeneity of bacterial populations in the Moselle River is shown and emphasized. Consequently, we recommend that replicate samples be taken on each occasion when conducting a sampling program for a stream pollution survey.