Estimation of heterozygosity for single-probe multilocus DNA fingerprints

In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.      

Effects of subtherapeutic administration of antimicrobial agents to beef cattle on the prevalence of antimicrobial resistance in Campylobacter jejuni and Campylobacter hyointestinalis

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (> or =94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.     

Contribution of subsurface peat to CO2 and CH4 fluxes in a neotropical peatland

Tropical peatlands play an important role in the global carbon cycling but little is known about factors regulating carbon dioxide (CO₂) and methane (CH₄) fluxes from these ecosystems. Here, we test the hypotheses that (i) CO₂ and CH₄ are produced mainly from surface peat and (ii) that the contribution of subsurface peat to net C emissions is governed by substrate availability. To achieve this, in situ and ex situ CO₂ and CH₄ fluxes were determined throughout the peat profiles under three vegetation types along a nutrient gradient in a tropical ombrotrophic peatland in Panama. The peat was also characterized with respect to its organic composition using ¹³C solid state cross‐polarization magic‐angle spinning nuclear magnetic resonance spectroscopy. Deep peat contributed substantially to CO₂ effluxes both with respect to actual in situ and potential ex situ fluxes. CH₄ was produced throughout the peat profile with distinct subsurface peaks, but net emission was limited by oxidation in the surface layers. CO₂ and CH₄ production were strongly substrate‐limited and a large proportion of the variance in their production (30% and 63%, respectively) was related to the quantity of carbohydrates in the peat. Furthermore, CO₂ and CH₄ production differed between vegetation types, suggesting that the quality of plant‐derived carbon inputs is an important driver of trace gas production throughout the peat profile. We conclude that the production of both CO₂ and CH₄ from subsurface peat is a substantial component of the net efflux of these gases, but that gas production through the peat profile is regulated in part by the degree of decomposition of the peat.     

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale

Key message

Cefotaxime (100 mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production.

Abstract

Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F₁ hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100 mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100 mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.     

Cutinolytic esterase activity of bacteria isolated from mixed-plant compost and characterization of a cutinase gene from Pseudomonas pseudoalcaligenes

The objective of the current study was to examine cutinolytic esterase (i.e., cutinase) activity by pseudomonads and bacteria isolated from mixed-plant compost. Approximately 400 isolates representing 52 taxa recovered from mixed-plant compost using cuticle baits, along with 117 pseudomonad isolates obtained from a culture collection (i.e., non-compost habitats), were evaluated. The ability of isolates to degrade the synthetic cutin polycaprolactone (PCL) was initially measured. Isolates from 23 taxa recovered from the compost degraded PCL. As well, isolates from 13 taxa of pseudomonads cleared PCL. Secondary screening measured esterase activity induced by the presence of apple cuticle using the chromogenic substrate p-nitrophenyl butyrate. Eighteen isolates representing four taxa (Alcaligenes faecalis , Bacillus licheniformis , Bacillus pumilus , and Pseudomonas pseudoalcaligenes) recovered from compost exhibited substantial esterase activity when grown with cuticle. In contrast, none of the pseudomonad isolates from the culture collection produced appreciable esterase activity. Although degradation of PCL was not correlated with esterase activity, isolates that were unable to degrade PCL failed to produce measureable esterase activities. Zymogram analysis indicated that the esterases produced by bacteria from compost ranged in size from 29 to 47 kDa. A gene from P. pseudoalcaligenes (cutA) was found to code for a cutin-induced esterase consisting of 302 amino acids and a theoretical protein size of 32 kDa. The enzyme was unique and was most closely related to other bacterial lipases (≤48% similarity).     

Evaluation of approaches for measuring taxonomic completeness of lake profundal macroinvertebrate assemblages

1. Benthic macroinvertebrates (MI) are commonly used to assess freshwater ecosystems with the reference condition approach. Such assessments necessitate control for natural community variation, either by categorical typologies or by predictive models that have been widely and successfully developed for running water biota but not previously for lake profundal invertebrates. 2. We evaluated four modelling techniques [multivariate regression tree (MRT), limiting environmental differences, nonparametric multiplicative regression (NPMR) and River Invertebrate Prediction And Classification System (RIVPACS) and the operative Finnish lake typology for assessing taxonomic completeness (observed‐to‐expected number of taxa, O/E) of profundal MI assemblages. We used data from 74 and 33 minimally disturbed reference lake basins for calibration and validation of the approaches, respectively, and 72 test basins subject to various anthropogenic pressures to evaluate sensitivity to detect impact. Either all predicted taxa (threshold probability of capture P_t = 0+) or only those predicted to be captured with ≥0.25 probability were used to calculate O/E. 3. With P_t = 0.25, all four modelling approaches were accurate (mean O/E = 0.966–1.053) but imprecise (SD of O/E = 0.279–0.304) in predicting the fauna actually observed in validation sites. All models were subtly more precise than a null model (mean 1.038, SD 0.343) or the typology (1.046, 0.327). The taxon‐specific NPMR model was slightly more precise than the other three models based on site groupings. 4. The O/E values correlated relatively weakly (r = 0.55–0.86) among the approaches, which thus produced contrasting lake‐specific assessments, despite their seemingly comparable performances. Indeed, typology, suggesting that MI assemblages were impaired in 56% of test sites, was more sensitive than the other approaches (26–46%) as an indicator of human‐induced deterioration. However, this greater ostensible sensitivity seemed to be biased, as lake morphometry, a main driver of natural community variation, remained uncontrolled by the typology. 5. Generally, our exercise illustrates the inconclusiveness of the common validation criteria for the assessment methods. The apparent poor predictability of the profundal fauna, irrespective of the method, may partly stem from large observation error, which could be alleviated by more intensive sampling. However, instead of an O/E‐taxa index, some other metric encompassing quantitative aspects might be preferable for assessing these species‐poor communities.     

Antibiotics conspicuously affect community profiles and richness, but not the density of bacterial cells associated with mucosa in the large and small intestines of mice

The influence of three antibiotics (bacitracin, enrofloxacin, and neomycin sulfate) on the mucosa-associated enteric microbiota and the intestines of mice was examined. Antibiotics caused conspicuous enlargement of ceca and an increase in overall length of the intestine. However, there were no pathologic changes associated with increased cecal size or length of the intestine. Conspicuous reductions in the richness of mucosa-associated bacteria and changes to community profiles within the small (duodenum, proximal jejunum, middle jejunum, distal jejunum, and ileum) and large (cecum, ascending colon, and descending colon) intestine occurred in mice administered antibiotics. Communities in antibiotic-treated mice were dominated by a limited number of Clostridium-like (i.e. clostridial cluster XIVa) and Bacteroides species. The richness of mucosa-associated communities within the small and large intestine increased during the 14-day recovery period. However, community profiles within the large intestine did not return to baseline (i.e. relative to the control). Although antibiotic administration greatly reduced bacterial richness, densities of mucosa-associated bacteria were not reduced correspondingly. These data showed that the antibiotics, bacitracin, enrofloxacin, and neomycin sulfate, administered for 21 days to mice did not sterilize the intestine, but did impart a tremendous and prolonged impact on mucosa-associated bacterial communities throughout the small and large intestine.     

A large-scale survey of genetic copy number variations among Han Chinese residing in Taiwan

Background

Winter migration of immature brown trout (Salmo trutta) into freshwater rivers has been hypothesized to result from physiologically stressful combinations of high salinity and low temperature in the sea.

Results

We sampled brown trout from two Danish populations entering different saline conditions and quantified expression of the hsp70 and Na/K-ATPases α 1b genes following acclimation to freshwater and full-strength seawater at 2°C and 10°C. An interaction effect of low temperature and high salinity on expression of both hsp70 and Na/K-ATPase α 1b was found in trout from the river entering high saline conditions, while a temperature independent up-regulation of both genes in full-strength seawater was found for trout entering marine conditions with lower salinities.

Conclusion

Overall our results support the hypothesis that physiologically stressful conditions in the sea drive sea-run brown trout into freshwater rivers in winter. However, our results also demonstrate intra-specific differences in expression of important stress and osmoregulative genes most likely reflecting adaptive differences between trout populations on a regional scale, thus strongly suggesting local adaptations driven by the local marine environment.     

Diversity and Distribution of Commensal Fecal Escherichia coli Bacteria in Beef Cattle Administered Selected Subtherapeutic Antimicrobials in a Feedlot Setting

Escherichia coli strains isolated from fecal samples were screened to examine changes in phenotypic and genotypic characteristics including antimicrobial susceptibility, clonal type, and carriage of resistance determinants. The goal of this 197-day study was to investigate the influence of administration of chlortetracycline alone (T) or in combination with sulfamethazine (TS) on the development of resistance, dissemination of defined strain types, and prevalence of resistance determinants in feedlot cattle. Inherent tetracycline resistance was detected in cattle with no prior antimicrobial exposure. Antimicrobial administration was not found to be essential for the maintenance of inherently ampicillin-resistant and tetracycline-resistant (Tet^r) E. coli in control animals; however, higher Tet^r E. coli shedding was observed in animals subjected to the two treatments. At day 0, high tetracycline (26.7%), lower sulfamethoxazole-tetracycline (19.2%), and several other resistances were detected, which by the finishing phase (day 197) were restricted to ampicillin-tetracycline (47.5%), tetracycline (31.7%), and ampicillin-tetracycline-sulfamethoxazole (20.8%) from both treated and untreated cattle. Among the determinants, bla_TEM1, tet(A), and sul2 were prevalent at days 0 and 197. Further, E. coli from day 0 showed diverse antibiogram profiles and strain types, which by the finishing phase were limited to up to three, irrespective of the treatment. Some genetically identical strains expressed different phenotypes and harbored diverse determinants, indicating that mobile genetic elements contribute to resistance dissemination. This was supported by an increased linked inheritance of ampicillin and tetracycline resistance genes and prevalence of specific strains at day 197. Animals in the cohort shed increasingly similar genotypes by the finishing phase due to animal-to-animal strain transmission. Thus, characterizing inherent resistance and propagation of cohort-specific strains is crucial for determining antimicrobial resistance in cattle.