Pig Manure Contamination Marker Selection Based on the Influence of Biological Treatment on the Dominant Fecal Microbial Groups

The objective of this study was to identify a microbial marker for pig manure contamination. We quantified the persistence of four dominant bacterial groups from the pig intestinal tract throughout manure handling at 10 livestock operations (including aerobic digestion) by using molecular typing. The partial 16S rRNA genes of Bacteroides-Prevotella, Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium group isolates were amplified and analyzed by capillary electrophoresis single-strand conformation polymorphism. The most dominant bacterial populations were identified by cloning and sequencing their 16S rRNA genes. The results showed that Bifidobacterium spp. and, to a lesser extent, members of the BSL group, were less affected by the aerobic treatment than either Eubacterium-Clostridiaceae or Bacteroides-Prevotella. Two Bifidobacterium species found in raw manure were still present in manure during land application, suggesting that they can survive outside the pig intestinal tract and also survive aerobic treatment. The 16S-23S rRNA internal transcribed spacer of one species, Bifidobacterium thermacidophilum subsp. porcinum, was sequenced, and a specific pair of primers was designed for its detection in the environment. With this nested PCR assay, this potential marker was not detected in samples from 30 bovine, 30 poultry, and 28 human fecal samples or in 15 urban wastewater effluents. As it was detected in runoff waters after spreading of pig manure, we propose this marker as a suitable microbial indicator of pig manure contamination.Brittany represents only 7% of France but is the main pig production area and hosts approximately 14 million fatteners per year. This high concentration of confined pig feeding has led to the overapplication of manure to soil, which contributes to water pollution. Physical and biological manure treatment processes have been developed to limit nitrogen and phosphorus pollution (5). As these treatments were not designed to eliminate microbial pollution, even treated manure can contain pathogenic microorganisms (27) and agricultural soils and water systems can thus potentially still be contaminated through surface runoff and seepage. As manure application can increase the number of pathogens in the soil (18), pig feces may represent a significant risk to human health in Brittany. Currently, the monitoring of bacteria to assess fecal contamination (Escherichia coli, fecal coliforms, and enterococci) does not differentiate contamination from pig slurry from pollution by other animals or humans. It is thus important to develop analytic tools to specifically detect this source of pollution.Many studies have already proposed potential markers for the detection of host-specific fecal pollution (2, 3, 8, 12-15, 20, 37, 38, 48, 49). Much of this research has concentrated on distinguishing human and animal sources of contamination (3, 8, 20, 30, 38). Some studies have focused on identifying individual sources of animal pollution and have described molecular markers for feces from ducks (13), chickens (37), bovines (2, 3, 49), or cervids (6). Biomarkers have been proposed for porcine fecal contamination but rarely for porcine manure, the bacterial composition of which differs from that of porcine feces (9). Molecular markers have been developed to target the 16S rRNA gene sequences of dominant Eubacteria (2, 14, 43, 48) or methanogenic Archaebacteria (54) of the pig intestinal tract, whereas Khatib et al. (29) targeted the STII toxin gene from enterotoxigenic E. coli. Among the dominant groups of pig fecal Eubacteria, which include Bacteroides-Prevotella, Eubacterium-Clostridiacea, Lactobacillus-Streptococcus (34, 45, 51, 58), and to a lesser extent Bifidobacterium (40), the Bacteroides-Prevotella group has been particularly well studied (14, 22, 44). This marker of pig feces was described by Okabe et al. (44), but their work was based on feces sampled from only two farms and the number of clones analyzed was low. Gourmelon et al. (22) also detected the presence of a specific marker of pig feces belonging to the Bacteroides-Prevotella group in five stored manure samples. Although these studies revealed the presence of specific markers in fecal samples and in the subsequent pig manure samples, they did not address the possible disappearance of these anaerobic bacteria during the storage or biological treatment of the manure.Due to the lack of data concerning the bacterial flora of manure, the aims of this study were (i) to compare the monitoring of the Bacteroides-Prevotella group with that of Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium throughout the biological manure treatment process and (ii) to search for a molecular marker among these groups of bacteria that was consistently present in the manure intended for land application. In the first part of this study, the persistence of the dominant bacteria throughout treatment was studied by using molecular typing, capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) (45) based on the analysis of the 16S rRNA genes. CE-SSCP is a fingerprinting technique in which single-stranded DNA fragments of the same length are separated based on the conformation of their secondary structure (23). The major advantages of this technique are its reproducibility between runs and its high resolution power with fewer false results than with denaturing gradient gel electrophoresis (25, 26).The second part of this article describes the relevance of the potential marker of pig manure (Bifidobacterium thermacidophilum subsp. porcinum) selected according to the results of the CE-SSCP profiles and the subsequent identification of dominant peaks of the CE-SSCP profiles. The specificity of this pig marker was then tested by assessing the host distribution in a selection of fecal, manure, and wastewater samples.     

Effects of tryptophan mutation on the deprotonation and reprotonation kinetics of the Schiff base during the photocycle of bacteriorhodopsin.

The rates of deprotonation and reprotonation of the protonated Schiff base (PSB) are determined during the photocycle of nine bacteriorhodopsin mutants in which Trp-10, 12, 80, 86, 137, 138, 182 and 189 are individually substituted by either phenylalanine or cysteine. Of all the mutants, the replacement of Trp-86, Trp-182, and Trp-189 by phenylalanine and Trp-137 by cysteine is found to significantly alter the rate of the deprotonation, but not that of the reprotonation process. As compared with ebR, the Trp-86 mutation dramatically increases the rate of deprotonation of the PSB while the Trp-182 mutation greatly decreases this rate. Temperature dependence studies on the rate constants of the deprotonation demonstrate that the different energetic and entropic effects of the mutation are responsible for the observed different kinetic behavior of the Trp-86 and Trp-182 mutants as compared with that of ebR. In the case of Trp-86 mutant, a large decrease in both energy and entropy of activation suggests that the mutation of this tryptophan residue opens up the protein structure as a result of eliminating the hydrogen-bonding group on its side chain by a phenylalanine substitution. A correlation is observed between the proton pumping yield and the relative amplitudes of the slow deprotonation component but not with rate constants of the rise or decay process at constant pH. These results are best discussed in terms of the heterogeneity model (with parallel cycle) rather than back reaction model.     

The retinylidene Schiff base counterion in bacteriorhodopsin

Previous studies of bacteriorhodopsin have indicated interactions between Asp-85, Asp-212, Arg-82, and the retinylidene Schiff base. The counterion environment of the Schiff base has now been further investigated by using single and double mutants of the above amino acids. Chromophore regeneration from bacterioopsin proceeds to a normal extent in the presence of a single aspartate or glutamate residue at position 85 or 212, whereas replacement of both charged amino acids in the mutant Asp-85----Asn/Asp-212----Asn abolishes the binding of retinal. This indicates that a carboxylate group at either residue 85 or 212 is required as counterion for formation and for stabilization of the protonated Schiff base. Measurements of the pKa of the Schiff base reveal reductions of greater than 3.5 units for neutral single mutants of Asp-85 but only decreases of less than 1.2 units for corresponding substitutions of Asp-212, relative to the wild type. Substitutions of Asp-85 show large red shifts in the absorption spectrum that are partially reversible upon addition of anions, whereas mutants of Asp-212 display minor red shifts or blue shifts. We conclude, therefore, that Asp-85 is the retinylidene Schiff base counterion in wild-type bacteriorhodopsin. In the mutant Asp-85----Asn/Asp-212----Asn formation of a protonated Schiff base chromophore is restored in the presence of salts. The spectral properties of the double mutant are similar to those of the acid-purple form of bacteriorhodopsin. Upon addition of salts the folded structure of wild-type and mutant proteins can be stabilized at low pH in lipid/detergent micelles. The data indicate that exogenous anions serve as surrogate counterions to the protonated Schiff base, when the intrinsic counterions have been neutralized by mutation or by protonation.     

Seasonal variations in antioxidant enzyme activity in ram seminal plasma

Seminal oxidative stress status is emerging as a significant prognostic tool in assisted reproductive technology. A dynamic interplay between pro- and anti-antioxidant substances in the ejaculate is essential. In this study, we determined seasonal changes in the activity of the antioxidant enzyme defense system comprising superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx) and catalase (CAT) in seminal plasma (SP) of mature Rasa Aragonesa rams. This breed corresponds to a local Spanish genotype with a short seasonal anoestrus between May and July. In addition, the activity of these enzymes was measured in protein fractions isolated from ram SP by exclusion chromatography. Total protein content in ram SP was higher during the breeding season (October-February) with a significantly higher value in first ejaculates. Antioxidant enzyme activities were higher during the non-breeding season (March-September). Comparing first and second ejaculates, SOD and CAT activities were higher in the first of all months. However, GR and GPx activities changed throughout the year. Thus, GR activity was higher in July and August in first ejaculates, this difference being significant in July (4.53 versus 2.37 nmol substrate/minmg protein, P<0.05). Conversely, GPx activity was significantly higher in September and November in second ejaculates (21.1 versus 6.81 and 10.91 versus 5.33, respectively, P<0.05). After SP fractionation by exclusion chromatography, GR activity was located in fractions 1 and 2 being irrelevant in the following peaks, and CAT activity was not detected all along the chromatographic profile. GPx and SOD activities were spread out along all fractions with a main peak in fractions 6 and 7. Given that these two fractions showed the greatest capacity to recover and prevent cold-shock membrane injury [Barrios B, Pérez-Pé R, Gallego M, Tato A, Osada J, Muino-Blanco T, Cebrián-Pérez JA. Seminal plasma proteins revert the cold-shock damage on ram sperm membrane. Biol Reprod 2000;63:1531-7, Barrios B, Fernández-Juan M, Muino-Blanco T, Cebrián-Pérez J. Immunocytochemical localization and biochemical characterization of two seminal plasma proteins that protect ram spermatozoa against cold shock. J Androl 2005;26:539-49], we could suggest that the protective effect might be, at least partially, due to the antioxidant enzyme activity.     

High functional diversity in deep‐sea fish communities and increasing intraspecific trait variation with increasing latitude

Variation in both inter‐ and intraspecific traits affects community dynamics, yet we know little regarding the relative importance of external environmental filters versus internal biotic interactions that shape the functional space of communities along broad‐scale environmental gradients, such as latitude, elevation, or depth. We examined changes in several key aspects of functional alpha diversity for marine fishes along depth and latitude gradients by quantifying intra‐ and interspecific richness, dispersion, and regularity in functional trait space. We derived eight functional traits related to food acquisition and locomotion and calculated seven complementary indices of functional diversity for 144 species of marine ray‐finned fishes along large‐scale depth (50–1200 m) and latitudinal gradients (29°–51° S) in New Zealand waters. Traits were derived from morphological measurements taken directly from footage obtained using Baited Remote Underwater Stereo‐Video systems and museum specimens. We partitioned functional variation into intra‐ and interspecific components for the first time using a PERMANOVA approach. We also implemented two tree‐based diversity metrics in a functional distance‐based context for the first time: namely, the variance in pairwise functional distance and the variance in nearest neighbor distance. Functional alpha diversity increased with increasing depth and decreased with increasing latitude. More specifically, the dispersion and mean nearest neighbor distances among species in trait space and intraspecific trait variability all increased with depth, whereas functional hypervolume (richness) was stable across depth. In contrast, functional hypervolume, dispersion, and regularity indices all decreased with increasing latitude; however, intraspecific trait variation increased with latitude, suggesting that intraspecific trait variability becomes increasingly important at higher latitudes. These results suggest that competition within and among species are key processes shaping functional multidimensional space for fishes in the deep sea. Increasing morphological dissimilarity with increasing depth may facilitate niche partitioning to promote coexistence, whereas abiotic filtering may be the dominant process structuring communities with increasing latitude.     

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.     

Dereplication of natural products from complex extracts by regression analysis and molecular networking: case study of redox-active compounds from <Emphasis Type="Italic">Viola alba</Emphasis> subsp. <Emphasis Type="Italic">dehnhardtii</Emphasis>

Introduction

In natural product research, bioassay-guided fractionation was previously widely employed but is now judged to be inadequate in terms of time and cost, particularly if only known compounds are ultimately isolated. The development of metabolomics, along with improvements in analytical tools, allows comprehensive metabolite profiling. This enables dereplication to target unknown active compounds early in the purification workflow.

Objectives

Starting from an ethanolic extract of violet leaves, this study aims to predict redox active compounds within a complex matrix through an untargeted metabolomics approach and correlation analysis.

Methods

Rapid fractionation of crude extracts was carried out followed by multivariate data analysis (MVA) of liquid chromatography–high resolution mass spectrometry (LC–HRMS) profiles. In parallel, redox active properties were evaluated by the capacity of the molecules to reduce 2,2-diphenyl-1-picrylhydrazyl (DPPH^·) and superoxide (O₂ ^·?) radicals using UV–Vis and electron spin resonance spectroscopies (ESR), respectively. A spectral similarity network (molecular networking) was used to highlight clusters involved in the observed redox activities.

Results

Dereplication on Viola alba subsp. dehnhardtii highlighted a reproducible pool of redox active molecules. Polyphenols, particularly O-glycosylated coumarins and C-glycosylated flavonoids, were identified and de novo dereplicated through molecular networking. Confirmatory analyses were undertaken by thin layer chromatography (TLC)–DPPH–MS assays and nuclear magnetic resonance (NMR) spectra of the most active compounds.

Conclusion

Our dereplication strategy allowed the screening of leaf extracts to highlight new biologically active metabolites in few steps with a limited amount of crude material and reduced time-consuming manipulations. This approach could be applied to any kind of natural extract for the study of various biological activities.

     

Epizootiological studies of Amblyospora camposi (Microsporidia: Amblyosporidae) in Culex renatoi (Diptera: Culicidae) and Paracyclops fimbriatus fimbriatus (Copepoda: Cyclopidae) in a bromeliad habitat

The epizootiology of Amblyospora camposi was studied in a natural population of Culex renatoi, a bromeliad-inhabiting mosquito, and its intermediate host, Paracyclops fimbriatus fimbriatus, over a 2-year period. Twenty Eryngium cabrerae plants were sampled monthly from January 2003 to January 2005 and the prevalence of A. camposi in P.f. fimbriatus and Cx. renatoi populations was determined. The monthly prevalence rates of meiospore infections in Cx. renatoi larvae never exceeded 5.5% and was detected in 50% of the monthly samples. Meiospores were available in plants over the course of the study at a mean concentration of 2 x 10(4) meiospores/ml. Within each plant the parasite was maintained by horizontal transmission. P.f. fimbriatus with vegetative stages and mature spores were found regularly in bromeliads suggesting efficient meiospore infectivity to field copepod populations. The mean concentration of spores from copepods found in plants was 8 x 10(2) spores/ml. Infections in copepods were detected in 54% of the monthly samples with a prevalence rate ranging from 0.55 to 17.4% and an overall average of 5.1%. Vegetative stages in fourth instar mosquito larvae (probably derived from the horizontal pathway via spores formed in copepods) were detected in 12.5% of the monthly samples with an overall prevalence rate of 1.1%. Infections in female and male adults were detected in 20.8% of the monthly samples with an overall average of 4.1% and 6.8%, respectively.