Phylogenetic analysis of Bacteroidales 16S rRNA gene sequences from human and animal effluents and assessment of ruminant faecal pollution by real‐time PCR

Aims: The aims of this study were to evaluate the host‐specific distribution of Bacteroidales 16S rRNA gene sequences from human‐ and animal‐related effluents and faeces, and to define a ruminant‐specific marker. Methods and Results: Bacteroidales 16S rRNA gene clone libraries were constructed from samples of effluent (sewage, bovine manure and pig slurry) and faeces (human, bovine, pig and wild bird), using PCR primers targeting order Bacteroidales. The phylogenetic analysis revealed six main distinct human‐, bovine‐, pig‐ and wild bird‐specific clusters. From the bovine‐specific cluster II, we designed a ruminant‐specific marker, Rum‐2‐Bac, and this showed 97% sensitivity (n = 30) and 100% specificity (n = 40) when tested by TaqMan^® real‐time PCR. Average concentrations of this marker in bovine and sheep faeces and in bovine manure were 8·2 ± 0·5, 8·4 ± 1·3 and 7 ± 0·5 log₁₀ copies per gram, respectively. It was also quantified in samples of runoff water impacted by bovine manure, with average concentrations of 5·1 ± 0·3 log₁₀ copies per millilitre water. Conclusions: Our results confirmed that some members of Bacteroidales isolated from effluents and faeces had host‐specific distributions. Identification of a bovine‐specific cluster made it possible to design a reliable ruminant‐specific marker. Significance and Impact of the Study: The host‐specific distribution of Bacteroidales sequences from effluents mirrored the host‐specific distribution of sequences observed in individual faeces. This efficient new ruminant‐specific Bacteroidales 16S rRNA marker represents a useful addition to the microbial source tracking toolbox.     

Relevance of bacteroidales and F-specific RNA bacteriophages for efficient fecal contamination tracking at the level of a catchment in France

The relevance of three host-associated Bacteroidales markers (HF183, Rum2Bac, and Pig2Bac) and four F-specific RNA bacteriophage genogroups (FRNAPH I to IV) as microbial source tracking markers was assessed at the level of a catchment (Daoulas, France). They were monitored together with fecal indicators (Escherichia coli and enterococci) and chemophysical parameters (rainfall, temperature, salinity, pH, and turbidity) by monthly sampling over 2 years (n = 240 water samples) and one specific sampling following an accidental pig manure spillage (n = 5 samples). During the 2-year regular monitoring, levels of E. coli, enterococci, total F-specific RNA bacteriophages, and the general Bacteroidales marker AllBac were strongly correlated with one another and with Rum2Bac (r = 0.37 to 0.50, P < 0.0001). Their correlations with HF183 and FRNAPH I and II were lower (r = 0.21 to 0.29, P < 0.001 to P < 0.0001), and HF183 and enterococci were associated rather than correlated (Fisher's exact test, P < 0.01). Rum2Bac and HF183 enabled 73% of water samples that had ≥ 2.7 log(10) most probably number (MPN) of E. coli/100 ml to be classified. FRNAPH I and II enabled 33% of samples at this contamination level to be classified. FRNAPH I and II complemented the water sample classification obtained with the two Bacteroidales markers by an additional 8%. Pig2Bac and FRNAPH III and IV were observed in a small number of samples (n = 0 to 4 of 245). The present study validates Rum2Bac and HF183 as relevant tools to trace fecal contamination originating from ruminant or human waste, respectively, at the level of a whole catchment.     

Kinetic model‐based prediction of the persistence of Salmonella enterica serovar Typhimurium under tropical agricultural field conditions

Aim: Present a kinetic model‐based approach for using isothermal data to predict the survival of manure‐borne enteric bacteria under dynamic conditions in an agricultural environment. Methods and Results: A model to predict the survival of Salmonella enterica serovar Typhimurium under dynamic temperature conditions in soil in the field was developed. The working hypothesis was that the inactivation phenomena associated with the survival kinetics of an organism in an agricultural matrix under dynamic temperature conditions is for a large part due to the cumulative effect of inactivation at various temperatures within the continuum registered in the matrix in the field. The modelling approach followed included (i) the recording of the temperature profile that the organism experiences in the field matrix, (ii) modelling the survival kinetics under isothermal conditions at a range of temperatures that were registered in the matrix in the field; and (iii) using the isothermal‐based kinetic models to develop models for predicting survival under dynamic conditions. The time needed for 7 log CFU g^?1Salmonella Typhimurium in manure and manure‐amended soil to reach the detection limit of the enumeration method (2 log CFU g^?1) under tropical conditions in the Central Agro‐Ecological Zone of Uganda was predicted to be 61–68 days and corresponded with observed CFU of about 2·2–3·0 log CFU g^?1, respectively. The Bias and Accuracy factor of the prediction was 0·71–0·84 and 1·2–1·4, respectively. Conclusions: Survival of Salm. Typhimurium under dynamic field conditions could be for 71–84% determined by the developed modelling approach, hence substantiating the working hypothesis. Significance and Impact of the Study: Survival kinetic models obtained under isothermal conditions can be used to develop models for predicting the persistence of manure‐borne enteric bacteria under dynamic field conditions in an agricultural environment.     

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems

Aims: This study was designed to evaluate the usefulness of quantification by real‐time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90‐431). Methods and Results: Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10⁵ GU l^?1) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57–100% of the samples. Conclusions: These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real‐time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. Significance and Impact of the Study: This study shows the possibility of using real‐time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.     

Determination of antimicrobial resistance and virulence gene signatures in surface water isolates of Escherichia coli

Aims: To determine the occurrence of Escherichia coli harbouring virulence markers of shiga‐ or entero‐toxins and resistance to antimicrobials in surface waters. Methods and Results: Surface water samples were collected at six locations of the river Gomti. E. coli isolates (n = 90) were characterized for their pathogenic potential using polymerase chain reaction to detect virulence genes as well as their sensitivity to antimicrobial agents using disc diffusion methods. In this study, 57·8% of E. coli isolates exhibited resistance to three or more antimicrobial agents. Sensitivity to cephotaxime, gentamicin and norfloxacin was observed in 7·8%, 48·9% and 77·8% of isolates, respectively. Both stx1 and stx2 genes were present in 15·6% of isolates while remaining isolates had either stx1 (17·8%) or stx2 (6·7%). The stx1 gene (33·3%) was more prevalent than stx2 (22·2%). The results indicate that the LT1 and ST1 genes were positive in 21·2% of isolates. Conclusions: The presence of multi‐drug resistance and virulence genes in E. coli isolated from surface water being used for domestic and recreational purposes may result in waterborne outbreaks. Significance and Impact of the Study: The data will be useful in monitoring surface waters for forecasting and management of waterborne outbreaks.     

Aquatic food‐web dynamics following incorporation of nutrients derived from Atlantic anadromous fishes

Changes in the isotopic composition (δ¹³C and δ¹⁵N) in biofilm, macro‐invertebrates and resident salmonids were used to characterize temporal dynamics of marine derived nutrients (MDNs) incorporation between stream reaches with and without MDN inputs. Five Atlantic rivers were chosen to represent contrasting MDN subsidies: four rivers with considerable numbers of anadromous fishes; one river with little MDN input. Rainbow smelt Osmerus mordax, alewife Alosa pseudoharengus, sea lamprey Petromyzon marinus and Atlantic salmon Salmo salar, were the primary anadromous species for the sampled rivers. Regardless of the spatial resolution or the pathway of incorporation, annual nutrient pulses from spawning anadromous fishes had a positive effect on isotopic enrichment at all trophic levels (biofilm, 1·2–5·4‰; macro‐invertebrates, 0·0–6·8‰; fish, 1·2–2·6‰). Community‐wide niche space shifted toward the marine‐nutrient source, but the total ecological niche space did not always increase with MDN inputs. The time‐integrated marine‐nutrient resource contribution to the diet of S. salar parr and brook trout Salvelinus fontinalis ranged between 16·3 and 36·0% during anadromous fish‐spawning periods. The high degree of spatio‐temporal heterogeneity in marine‐nutrient subsidies from anadromous fishes lead to both direct and indirect pathways of MDN incorporation into stream food webs. This suggests that organisms at many trophic levels derive a substantial proportion of their energy from marine resources when present. The current trend of declining anadromous fish populations means fewer nutrient‐rich marine subsidies being delivered to rivers, diminishing the ability to sustain elevated riverine productivity.     

Phenotypic and genotypic bacterial antimicrobial resistance in liquid pig manure is variously associated with contents of tetracyclines and sulfonamides

Aims: Antibiotic residues as well as antibiotic‐resistant bacteria in environmental samples might pose a risk to human health. This study aimed to investigate the association between antibiotic residues and bacterial antimicrobial resistance in liquid pig manure used as fertilizer. Methods and Results: Concentrations of tetracyclines (TETs) and sulfonamides (SULs) were determined by liquid chromatography‐mass spectrometry in 305 pig manure samples; antibiotic contents were correlated to the phenotypic resistance of Escherichia coli (n = 613) and enterococci (n = 564) towards up to 24 antibiotics. In 121 samples, the concentration of the TET resistance genes tet(M), tet(O) and tet(B) was quantified by real‐time‐PCR. TETs were found in 54% of the samples. The median sum concentration of all investigated TETs in the positive samples was 0·73 mg kg^?1. SULs were found with a similar frequency (51%) and a median sum concentration of 0·15 mg kg^?1 in the positive samples. Associated with the detection of TETs and/or SULs, resistance rates were significantly elevated for several substances – some of them not used in farm animals, e.g. chloramphenicol and synercid. In addition, multiresistant isolates were found more often in samples containing antibiotics. Analysis of the resistance genes tet(M) and tet(O) already showed a significant increase in their concentrations – but not in tet(B) – in the lowest range of total TET concentration. Mean tet(M) concentrations increased by the factor of 4·5 in the TET concentration range of 0·1–1 mg kg^?1, compared to negative manure samples. Conclusions: Antibiotic contamination of manure seems to be associated with a variety of changes in bacterial resistance, calling for a prudent use of antibiotics in farm animals. Significance and Impact of the Study: This study provides an interdisciplinary approach to assess antimicrobial resistance by combining the microbiological analysis of bacterial resistance with high quality chemical analysis of antibiotic residues in a representative number of environmental samples.     

Modelling the future hydroclimatology of the lower Fraser River and its impacts on the spawning migration survival of sockeye salmon

Short episodic high temperature events can be lethal for migrating adult Pacific salmon (Oncorhynchus spp.). We downscaled temperatures for the Fraser River, British Columbia to evaluate the impact of climate warming on the frequency of exceeding thermal thresholds associated with salmon migratory success. Alarmingly, a modest 1.0 °C increase in average summer water temperature over 100 years (1981–2000 to 2081–2100) tripled the number of days per year exceeding critical salmonid thermal thresholds (i.e. 19.0 °C). Refined thresholds for two populations (Gates Creek and Weaver Creek) of sockeye salmon (Oncorhynchus nerka) were defined using physiological constraint models based on aerobic scope. While extreme temperatures leading to complete aerobic collapse remained unlikely under our warming scenario, both populations were increasingly forced to migrate upriver at reduced levels of aerobic performance (e.g. in 80% of future simulations, ≥90% of salmon encountered temperatures exceeding population‐specific thermal optima for maximum aerobic scope; T_opt=16.3 °C for Gates Creek and T_opt=14.5 °C for Weaver Creek). Assuming recent changes to river entry timing persist, we also predicted dramatic increases in the probability of freshwater mortality for Weaver Creek salmon due to reductions in aerobic, and general physiological, performance (e.g. in 42% of future simulations≥50% of Weaver Creek fish exceeded temperature thresholds associated with 0–60% of maximum aerobic scope). Potential for adaptation via directional selection on run‐timing was more evident for the Weaver Creek population. Early entry Weaver Creek fish experienced 25% (range: 15–31%) more suboptimal temperatures than late entrants, compared with an 8% difference (range: 0–17%) between early and late Gates Creek fish. Our results emphasize the need to consider daily temperature variability in association with population‐specific differences in behaviour and physiological constraints when forecasting impacts of climate change on migratory survival of aquatic species.     

Population structure of the olive flounder (Paralichthys olivaceus) in Korea inferred from microsatellite marker analysis

The population structure of olive flounder Paralichthys olivaceus was estimated using nine polymorphic microsatellite (MS) loci in 459 individuals collected from eight populations, including five wild and three hatchery populations in Korea. Genetic variation in hatchery (mean number of alleles per locus, A = 10·2–12·1; allelic richness, A_R = 9·3–10·1; observed heterozygosity, H_O = 0·766–0·805) and wild (mean number of alleles per locus, A = 11·8–19·6; allelic richness, A_R = 10·9–16·1; observed heterozygosity, H_O = 0·820–0·888) samples did not differ significantly, suggesting a sufficient level of genetic variation in these well‐managed hatchery populations, which have not lost a substantial amount of genetic diversity. Neighbour‐joining tree and principal component analyses showed that genetic separation between eastern and pooled western and southern wild populations in Korea was probably influenced by restricted gene flow between regional populations due to the barrier effects of sea currents. The pooled western and southern populations are genetically close, perhaps because larval dispersal may depend on warm currents. One wild population (sample from Wando) was genetically divergent from the main distribution, but it was genetically close to hatchery populations, indicating that the genetic composition of the studied populations may be affected by hydrographic conditions and the release of fish stocks. The estimated genetic population structure and potential applications of MS markers may aid in the proper management of P. olivaceus populations.     

Microsatellite variation and population genetic structure of the red throat emperor on the Great Barrier Reef

Analysis (using three analytical approaches) of eight microsatellite markers from six locations in three geographic regions of the Great Barrier Reef (GBR), including populations that differed in demographic characteristics, showed no evidence of genetic stock structure in the red throat emperor Lethrinus miniatus. Measures of inter‐population differentiation were non‐significant (P ≥ 0·67). Using a Bayesian clustering approach, ‘admixture’ was detected (mean alpha values >1) with allele frequencies for each of the locations sampled being correlated equally with allele frequencies from all locations sampled. The number of populations (K) identified was one, based on the estimates of the probability of the data at various K values (K = 1, 2, 3, … 6). Additionally, alpha values did not stabilize to relatively constant values in any of the Bayesian analyses performed, indicating that there was no real genetic structure between locations. Analysis of genetic variation as detected by analysis of molecular variance (AMOVA) indicated that almost all of the variance in the data (99·74%, P ≤ 0·023) was within populations, rather than among populations (0·15%, P ≤ 0·176) or amongst regions sampled (0·10%, P ≤ 0·247) on the GBR. F_st statistics identified four individual loci having statistically significant differentiation among populations, but these were only related to one out of 12 pair‐wise comparisons where populations differed demographically. Given these results (albeit using neutral markers), together with the capacity of adults and larvae to be mobile between reefs on the inter‐connected GBR, it is considered unlikely that L. miniatus populations exist as distinct genetic stocks in the GBR. It is therefore not possible, using neutral markers, to reject the null hypothesis that the fishery be managed as a single panmictic stock.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure and manure‐amended soil under tropical climatic conditions in Sub‐Saharan Africa

Aims: To establish the fate of Escherichia coli O157:H7 and Salmonella Typhimurium in manure and manure‐amended agricultural soils under tropical conditions in Sub‐Saharan Africa. Methods and Results: Survival of nonvirulent E. coli O157:H7 and Salm. Typhimurium at 4 and 7 log CFU g^?1 in manure and manure‐amended soil maintained at ≥80% r.h. or exposed to exclusive field or screen house conditions was determined in the Central Agro‐Ecological Zone of Uganda. Maintaining the matrices at high moisture level promoted the persistence of high‐density inocula and enhanced the decline of low‐density inocula in the screen house, but moisture condition did not affect survival in the field. The large majority of the survival kinetics displayed complex patterns corresponding to the Double Weibull model. The two enteric bacteria survived longer in manure‐amended soil than in manure. The 7 log CFU g^?1E. coli O157:H7 and Salm. Typhimurium survived for 49–84 and 63–98 days, while at 4 log CFU g^?1, persistence was 21–28 and 35–42 days, respectively. Conclusions: Under tropical conditions, E. coli O157:H7 and Salm. Typhimurium persisted for 4 and 6 weeks at low inoculum density and for 12 and 14 weeks at high inoculum density, respectively. Significance and Impact of the Study: Persistence in the tropics was (i) mostly shorter than previously observed in temperate regions thus suggesting that biophysical conditions in the tropics might be more detrimental to enteric bacteria than in temperate environments; (ii) inconsistent with published data isothermally determined previously hence indicating the irrelevance of single point isothermal data to estimate survival under dynamic temperature conditions.     

Interaction between migration behaviour and estuarine mortality in cultivated Atlantic salmon Salmo salar smolts

Migration behaviour and estuarine mortality of cultivated Atlantic salmon Salmo salar smolts in a 16 km long estuary were studied using two methods: (1) acoustic telemetry and (2) group tagging in combination with trap nets. Progression rates of surviving individuals through the estuary were relatively slow using both methods [0·38 L_T (total length) s^?1 v. 0·25 L_T s^?1]. In 2012, the progression rate was slow from the river to the estuary (0·55 L_T s^?1) and the first part of the estuary (0·31 L_T s^?1), but increased thereafter (1·45–2·21 L_T s^?1). In 2013, the progression rate was fast from the river to the estuary (4·31 L_T s^?1) but was slower thereafter (0·18–0·91 L_T s^?1). Survival to the fjord was higher in 2012 (47%) compared to 2013 (6%). Fast moving individuals were more likely to migrate successfully through the estuary compared to slower moving individuals. Adult recapture of coded‐wire‐tagged S. salar was generally low (0·00–0·04%). Mortality hot spots were related to topographically distinct areas such as the river outlet (in 2012) or the sill separating the estuary and the fjord (in 2013). At the sill, an aggregation of cod Gadus morhua predating on cultivated smolts was identified. The results indicate that slow progression rates through the estuary decreases the likelihood of smolts being detected outside the estuary. The highly stochastic and site‐specific mortality patterns observed in this study highlight the complexity in extrapolating mortality patterns of single release groups to the entire smolt run of wild S. salar.     

Catch composition,reproductive biology and diet of the bramble shark Echinorhinus brucus (Squaliformes: Echinorhinidae) from the south‐eastern Arabian Sea

Fishery and biological data are presented for the poorly known bramble shark Echinorhinus brucus (Squaliformes: Echinorhinidae), from the deep waters of the south‐eastern Arabian Sea. A total of 5318 individuals from by‐catch landings of deep‐water bottom set longlines, gillnets and shrimp trawl fisheries operating at depths of 200–1200 m were recorded between January 2008 and December 2011 at the Kochi Fisheries Harbour (Kerala). A total of 431 individuals, from 46 to 318 cm total length (L_T) and 0·8 to 132 kg total mass (M_T), were examined to determine biological data for this species. The L_T at which 50% were mature (L_T50) for females and males was estimated at 189 and 187 cm L_T. Litter size ranged from 10 to 36 and size at birth was between 42 and 46 cm L_T. Dietary analysis of stomach contents revealed E. brucus feeds on a variety of prey including crustaceans (69% index of relative importance, I_RI), teleosts (25·8% I_RI), cephalopods (1·7% I_RI) and elasmobranchs (0·7% I_RI). This study provides the first detailed biological data for this species and also highlights the extent of the by‐catch fishery for this species in Indian waters.     

Persistence and diversity of faecal coliform and enterococci populations in faecally polluted waters

Aim: To assess the persistence and diversity of faecal bacterial populations (faecal coliforms and enterococci) that have recently been included in microbial source tracking (MST) predictive models. Methods and Results: The analysed bacterial populations included members of the enterococci group (ENT) [Enterococcus faecium (FM), Enterococcus faecalis (FS) and Enterococcus hirae (HIR)] and the faecal coliform group (FC) [diverse Escherichia coli phenotypes (ECP) and cellobiose‐negative faecal coliforms (CNFC)]. The inactivation of these distinct groups was monitored over time on‐site in river by biochemical fingerprinting, and diversity indices were calculated. Among the different analysed species belonging to the ENT group, HIR persisted longer and was able to replicate in the environment at a higher rate. On the other hand, ECP and NCFC showed a similar persistence throughout the different seasons. The diversity index (Di) for FC increased substantially in the summer after 96 h to a maximum value of 0·96. On the other hand, the Di for ENT diminished over the same period to a value of 0·86, suggesting a different persistence for the different species integrating this group. Conclusions: The persistence of ECP, CNFC, FM and FS in the aquatic environment is high, particularly for the members of the FC and in the summer season. On the contrary, HIR is able to replicate in the environment at a high rate even in winter, and therefore, its inclusion in MST predictive models is discouraged. Significance and Impact of the Study: ECP, CNFC, FMFS and HIR have been proposed as additional variables in MST predictive models. However, the different persistence of HIR compared with the other variables should be taken into account for the development of such models.     

Evaluation of Lactobacillus sobrius/L. amylovorus as a New Microbial Marker of Pig Manure

Based on a comparison of the dominant microbial populations in 17 pig manure samples and using a molecular typing method, we identified a species, Lactobacillus sobrius and Lactobacillus amylovorus (which now are considered a single species and are designated L. sobrius/amylovorus here), that was consistently found in manure. The aim of the present study was to confirm by real-time PCR the relevance of this species as a marker of pig fecal contamination. The specificity of L. sobrius/amylovorus was evaluated in human and animal DNA extracted from feces. The real-time PCR assay then was applied to water samples, including effluents from urban wastewater treatment plants, runoff water, and rivers. L. sobrius/amylovorus was consistently present in all samples of swine origin: 48 fecal samples, 18 from raw manure and 10 from biologically treated manure at mean concentrations of 7.2, 5.9, and 5.0 log₁₀ cells/g, respectively. The species was not detected in any of the other livestock feces (38 samples from cattle and 16 from sheep), in the 27 human fecal samples, or in the 13 effluent samples from urban wastewater treatment plants. Finally, L. sobrius/amylovorus was not detected in runoff water contaminated by cattle slurry, but it was quantified at concentrations ranging from 3.7 to 6.5 log₁₀ cells/100 ml in runoff water collected after pig manure was spread on soil. Among the stream water samples in which cultured Escherichia coli was detected, 23% tested positive for L. sobrius/amylovorus. The results of this study indicate that the quantification of L. sobrius/amylovorus using real-time PCR will be useful for identifying pig fecal contamination in surface waters.Pig manure may contain pathogenic microorganisms that are harmful to humans and animals (11). These pathogens, which include bacteria, viruses, and protozoans, can survive for several weeks during the storage of manure and in the soil after manure is spread on the land (30). As a consequence, the large amount of manure that is produced and applied on land in many agricultural areas may impact water quality. It contributes to non-point source pollution, which is due partially to runoff from manured soil, especially when manure is spread just before rainfall. It is difficult to determine the origin of diffuse pollution, as it cannot be traced to a specific spot. Fecal indicators (Escherichia coli, fecal coliforms, and enterococci), which are commonly used to quantify fecal pollution, are present in the intestinal tracts of both humans and warm-blooded animals and thus cannot be used to distinguish contamination by pig manure from other sources of pollution. For this reason, alternative microbial indicators have been proposed for the identification of specific pollution sources.During the past 10 years, a few library-independent methods have been developed for the identification of pig fecal contamination. They are based mostly on the PCR amplification of specific genes or sequences, such as the STII toxin gene from enterotoxigenic E. coli (16), the internal transcribed spacer (ITS) sequence from Bifidobacterium thermacidophilum subsp. porcinum (26), the 16S rRNA gene of Bacteroides-Prevotella (5, 27, 31), and the methyl coenzyme M reductase gene from a methanogenic Archaea member (41). However, some of these methods are only qualitative, like the detection of B. thermacidophilum subsp. porcinum or of the mcrA and STII toxin genes, and do not allow the level of contamination to be quantified. Furthermore, it is noteworthy that the archaeal mcrA gene was not detected in 16% of the pig feces analyzed (41), and that the presence of the STII toxin gene depends on the level of E. coli in the sample, which needs to be greater than 100 cells to avoid false positives (16). Okabe et al. (31) quantified a Bacteroides-Prevotella pig-specific marker (Pig-Bac2) in water samples using real-time PCR. However, this marker lacks specificity, as the Pig-Bac2 marker also was present in human and cow feces at a concentration of 7 and 8 log₁₀ copies per g, respectively (31). Only one pig-specific Bacteroidales 16S rRNA gene marker (Pig-2-Bac), which was developed recently by Mieszkin et al. (27) using real-time PCR, appears to be suitable to quantify pig fecal contamination. However, one limit of targeting the Bacteroidales group could be their strictly anaerobic metabolism, which may influence their persistence in well-oxygenated water. Savichtcheva et al. (36) thus have reported that oxygen has a negative effect on the survival rate of Bacteroides fragilis. We thus consider it important to study biomarkers that are less sensitive to oxygen in order to extend the choice of tools for tracking sources of pollution by manure. Moreover, in the case of the downgrading of bathing or shellfish areas, when health and economic risks are involved, it could be useful to combine multiple markers to identify the source of pollution with certainty.In the search for potential pig manure markers, we recently analyzed the dominant bacterial groups of 17 raw pig manure samples using 16S rRNA-targeted PCR and the CE-SSCP (capillary electrophoresis-single-strand conformation polymorphism) molecular typing method (26). Among the dominant bacterial groups (Bacteroidales, Bifidobacterium, Eubacterium-Clostridiaceae, and Bacillus-Streptococcus-Lactobacillus), we highlighted the presence of a microaerophilic species, Lactobacillus sobrius, which was isolated from piglet feces previously (19). Lactobacilli are known to establish a stable population in the intestinal tract of piglets soon after birth (28, 39) and to rapidly become a dominant population of their intestinal flora, at least in the first days after weaning (2, 14, 34). Their concentration in pig feces has been estimated at about 3 × 10⁸ bacteria/g (9). Because of their protective effect against diarrhea, some species of Lactobacillus, including L. sobrius, particularly have been studied (20, 35). Konstantinov et al. (21) therefore designed a primer pair that specifically amplifies a fragment of the L. sobrius genome using real-time PCR. Finally, Jakava-Viljanen et al. (13) recently demonstrated very high similarity between the L. sobrius and L. amylovorus type and reference strains and representative porcine isolates based on their 16S rRNA gene sequence analysis. According to these results, L. sobrius and L. amylovorus constitute a single species and consequently are referred to as L. sobrius/amylovorus in this paper.Given the abundance of L. sobrius/amylovorus in piglet feces (19, 37) and its systematic presence in raw manure (26), we tested this species as a new marker of pig fecal contamination. The aims of our study were (i) to confirm the specificity of L. sobrius/amylovorus to pig feces by analyzing five host groups (human, pig, cattle, poultry, and sheep), manure and by-products of manure treatment, runoff water, and urban wastewaters, and (ii) to estimate the suitability of this marker to identify pig fecal contamination found in surface waters. The concentrations of L. sobrius/amylovorus were estimated by real-time PCR using the primers designed by Konstantinov et al. (21). They were compared to the levels of E. coli, total lactobacilli, and, for river water samples, to the concentrations of the pig-specific Bacteroidales 16S rRNA genetic marker (Pig-2-Bac) developed by Mieszkin et al. (27).     

Population origin of Atlantic sturgeon Acipenser oxyrinchus oxyrinchus by‐catch in U.S. Atlantic coast fisheries

Microsatellite DNA and mitochondrial DNA control‐region sequence analyses were used to determine the population and distinct population segment (DPS) origin of 173 Atlantic sturgeon Acipenser oxyrinchus oxyrinchus encountered from the Gulf of Maine to Cape Hatteras, North Carolina, in NOAA's Northeast Fisheries Observer Program. It was found that the Hudson River was by far the greatest contributor to this coastal by‐catch, with 42·2–46·3% of specimens originating there. Generally, specimens represented the geographic province of the river in which they were spawned, but some specimens, particularly those originating in the South Atlantic DPS, moved to great distances. Genetic mixed‐stock analyses provide an accurate approach to determine the DPS and population origin of A. o. oxyrinchus by‐catch in coastal waters, but most informative management requires that these results be partitioned by locale, season, target fishery and gear type.     

The effect of catch‐and‐release angling at high water temperatures on behaviour and survival of Atlantic salmon Salmo salar during spawning migration

In this study, behaviour and survival following catch‐and‐release (C&R) angling was investigated in wild Atlantic salmon Salmo salar (n = 75) angled on sport fishing gear in the River Otra in southern Norway at water temperatures of 16·3–21·1° C. Salmo salar were tagged externally with radio transmitters and immediately released back into the river to simulate a realistic C&R situation. The majority of S. salar (91%) survived C&R. Most S. salar that were present in the River Otra during the spawning period 3–4 months later were located at known spawning grounds. Downstream movements (median furthest position: 0·5 km, range: 0·1–11·0 km) during the first 4 days after release were recorded for 72% of S. salar, presumably stress‐induced fallback associated with C&R. Individuals that fell back spent a median of 15 days before commencing their first upstream movement after release, and 34 days before they returned to or were located above their release site. Mortality appeared to be somewhat elevated at the higher end of the temperature range (14% at 18–21° C), although sample sizes were low. In conclusion, C&R at water temperatures up to 18° C had small behavioural consequences and was associated with low mortality (7%). Nevertheless, low levels of mortality occur due to C&R angling and these losses should be accounted for by management authorities in rivers where C&R is practised. Refinement of best practices for C&R may help to reduce mortality, particularly at warmer temperatures.     

Evaluation of methodology for detection of human adenoviruses in wastewater,drinking water,stream water and recreational waters

Aims: This study evaluates dialysis filtration and a range of PCR detection methods for identification and quantification of human adenoviruses in a range of environmental waters. Methods and Results: Adenovirus was concentrated from large volumes (50–200 l) of environmental and potable water by hollow fibre microfiltration using commercial dialysis filters. By this method, an acceptable recovery of a seeded control bacteriophage MS2 from seawater (median 95·5%, range 36–98%, n = 5), stream water (median 84·7%, range 23–94%, n = 5) and storm water (median 59·5%, range 6·3–112%, n = 5) was achieved. Adenovirus detection using integrated cell culture PCR (ICC‐PCR), direct PCR, nested PCR, real‐time quantitative PCR (qPCR) and adenovirus group F‐specific direct PCR was tested with PCR products sequenced for confirmation. Adenovirus was routinely detected from all water types by most methods, with ICC‐PCR more sensitive than direct‐nested PCR or qPCR. Group F adenovirus dominated in wastewater samples but was detected very infrequently in environmental waters. Conclusions and Implications: Human adenoviruses (HAdv) proved relatively common in environmental and potable waters when assessed using an efficient concentration method and sensitive detection method. ICC‐PCR proved most sensitive, could be used semiquantitatively and demonstrated virus infectivity but was time consuming and expensive. qPCR provided quantitative results but was c. ten‐fold less sensitive than the best methods.     

Incidence of Vibrio vulnificus in estuarine waters of the south Texas Coastal Bend region

Aims: To determine the occurrence of the human pathogen, Vibrio vulnificus, in south Texas coastal waters. Methods and Results: Coastal waters were sampled monthly between August 2006 and July 2007. Water temperature, dissolved oxygen, pH, salinity, conductivity and turbidity were measured during each sampling event. Culture‐based techniques utilizing Vibrio vulnificus agar (VVA) and membrane‐Enterococcus indoxyl‐β‐d ‐glucoside agar (mEI) were used to assess the occurrence and levels of V. vulnificus and the faecal contamination indicator group, enterococci, respectively. Vibrio vulnificus isolates were confirmed using colony‐blot hybridization with the species‐specific VVAP probe. Vibrio vulnificus was isolated at all sites throughout the year even when the water temperature dropped to 9·71°C. Significant correlations were found between concentrations of V. vulnificus and the abiotic factors, water temperature (P = 0·002) and dissolved oxygen (P = 0·028), as well as between concentrations of V. vulnificus and enterococci (P < 0·001). Conclusions: This study demonstrated the year‐round presence of V. vulnificus in coastal waters of south Texas. Significance and Impact of the Study: These findings indicate that the potential for human exposure to the pathogen, V. vulnificus, exists throughout the year. It also suggests that routinely monitored data might be used to predict the occurrence of the pathogen.     

Assessing the faecal source sensitivity and specificity of ruminant and human genetic microbial source tracking markers in the central Ethiopian highlands

This study tested genetic microbial source tracking (MST) methods for identifying ruminant- (BacR) and human-associated (HF183/BacR287, BacHum) bacterial faecal contaminants in Ethiopia in a newly created regional faecal sample bank (n = 173). BacR performed well, and its marker abundance was high (100% sensitivity (Sens), 95% specificity (Spec), median log₁₀ 8·1 marker equivalents (ME) g⁻¹ ruminant faeces). Human-associated markers tested were less abundant in individual human samples (median: log₁₀ 5·4 and 4·2 (ME + 1) g⁻¹) and were not continuously detected (81% Sens, 91% Spec for BacHum; 77% Sens, 91% Spec for HF183/BacR287). Furthermore, the pig-associated Pig2Bac assay was included and performed excellent (100% Sens, 100% Spec). To evaluate the presence of MST targets in the soil microbiome, representative soil samples were tested during a whole seasonal cycle (n = 60). Only BacR could be detected, but was limited to the dry season and to sites of higher anthropogenic influence (log₁₀ 3·0 to 4·9 (ME + 1) g⁻¹ soil). In conclusion, the large differences in marker abundances between target and non-target faecal samples (median distances between distributions ≥log₁₀ 3 to ≥log₁₀ 7) and their absence in pristine soil indicate that all tested assays are suitable candidates for diverse MST applications in the Ethiopian area.