Ovine pedomics: the first study of the ovine foot 16S rRNA-based microbiome

We report the first study of the bacterial microbiome of ovine interdigital skin based on 16S rRNA by pyrosequencing and conventional cloning with Sanger-sequencing. Three flocks were selected, one a flock with no signs of footrot or interdigital dermatitis, a second flock with interdigital dermatitis alone and a third flock with both interdigital dermatitis and footrot. The sheep were classified as having either healthy interdigital skin (H) and interdigital dermatitis (ID) or virulent footrot (VFR). The ovine interdigital skin bacterial community varied significantly by flock and clinical condition. The diversity and richness of operational taxonomic units was greater in tissue from sheep with ID than H or VFR-affected sheep. Actinobacteria, Bacteriodetes, Firmicutes and Proteobacteria were the most abundant phyla comprising 25 genera. Peptostreptococcus, Corynebacterium and Staphylococcus were associated with H, ID and VFR, respectively. Sequences of Dichelobacter nodosus, the causal agent of ovine footrot, were not amplified because of mismatches in the 16S rRNA universal forward primer (27F). A specific real-time PCR assay was used to demonstrate the presence of D. nodosus, which was detected in all samples including the flock with no signs of ID or VFR. Sheep with ID had significantly higher numbers of D. nodosus (10⁴–10⁹ cells per g tissue) than those with H or VFR feet.     

Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition

Background

Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.

Methodology/Principal Findings

We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.

Conclusions

M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×10⁵ cells g⁻¹ using Griffiths and 4.25×10⁶ cells g⁻¹ using FastDNA® Spin kit.     

LCAT deficiency in mice is associated with a diminished adrenal glucocorticoid function

In vitro studies have suggested that HDL and apoB-containing lipoproteins can provide cholesterol for synthesis of glucocorticoids. Here we assessed adrenal glucocorticoid function in LCAT knockout (KO) mice to determine the specific contribution of HDL-cholesteryl esters to adrenal glucocorticoid output in vivo. LCAT KO mice exhibit an 8-fold higher plasma free cholesterol-to-cholesteryl ester ratio (P < 0.001) and complete HDL-cholesteryl ester deficiency. ApoB-containing lipoprotein and associated triglyceride levels are increased in LCAT KO mice as compared with C57BL/6 control mice (44%; P < 0.05). Glucocorticoid-producing adrenocortical cells within the zona fasciculata in LCAT KO mice are devoid of neutral lipids. However, adrenal weights and basal corticosterone levels are not significantly changed in LCAT KO mice. In contrast, adrenals of LCAT KO mice show compensatory up-regulation of genes involved in cholesterol synthesis (HMG-CoA reductase; 516%; P < 0.001) and acquisition (LDL receptor; 385%; P < 0.001) and a marked 40–50% lower glucocorticoid response to adrenocorticotropic hormone exposure, endotoxemia, or fasting (P < 0.001 for all). In conclusion, our studies show that HDL-cholesteryl ester deficiency in LCAT KO mice is associated with a 40–50% lower adrenal glucocorticoid output. These findings further highlight the important novel role for HDL as cholesterol donor for the synthesis of glucocorticoids by the adrenals.     

Short-term in vivo inhibition of nitric oxide synthase with L-NAME influences the contractile function of single left ventricular myocytes in rats

The main purpose of this study was to investigate the effects of short-term L-NAME treatment on the contractile function of left ventricle (LV) myocytes and the expression of proteins related to Ca(2+) homeostasis. Data from Wistar rats treated with L-NAME (L group, n = 20; 0.7?g/L in drinking water; 7?days) were compared with results from untreated controls (C group, n = 20). Cardiomyocytes from the L group showed increased (p?< 0.05) fractional shortening (23%) and maximum rate of shortening (20%) compared with the C group. LV from the L group also showed increased (p?< 0.05) expression of the ryanodine receptor 2 and Na(+)/Ca(2+) exchanger proteins (76% and 83%, respectively; p?< 0.05). However, the L and C groups showed similar in vivo hemodynamic parameters of cardiac function. In conclusion, short-term NOS inhibition determines an increased expression of Ca(2+) regulatory proteins, which contributes to improving cardiomyocyte contractile function, preserving left ventricular function.     

Traits reveal how habitat‐quality gradients structure small mammal communities in a fragmented tropical landscape

Understanding the relationships among community structure, vegetation structure and availability of food resources are a key to unravelling the ecological processes that structure biological communities. In this study, we tested (i) whether the composition of small mammal communities changed across gradients in habitat quality in tropical forest fragments, and (ii) whether any observed change could be explained by the functional traits of species. We sampled 24 trapping grids in fragments of semi‐deciduous forest, in each of two 6‐month periods. We considered each trapping grid as a sampling unit, for which we collected three datasets: an environmental matrix (vegetation structure and food resource availability), the abundance of small mammal species (community structure) and a matrix of functional traits (ecological and morphological traits which express tolerance to habitat disturbance and trophic guild). We used an RLQ approach to evaluate the association between traits and environmental gradients. Forest‐specialist and scansorial–arboreal species were associated with more complex habitat that had greater litter and canopy cover and more fallen logs. In relation to trophic guilds, granivore (fruit seeds), insectivorous and omnivorous species were also associated with higher complexity habitat, while frugivores were associated with shrub cover and availability of fruits. We conclude that functional traits (habitat use, use of vertical strata and diet) provide valuable insights into the distribution of small mammals along gradients of habitat quality in tropical forest fragments. We highlight that communities studies in fragmented landscapes should investigate the different components of biodiversity not only in landscape‐scale but also in habitat scale. Abstract in Portuguese is available with online material.     

A Multifactor Analysis of Fungal and Bacterial Community Structure in the Root Microbiome of Mature Populus deltoides Trees

Bacterial and fungal communities associated with plant roots are central to the host health, survival and growth. However, a robust understanding of the root-microbiome and the factors that drive host associated microbial community structure have remained elusive, especially in mature perennial plants from natural settings. Here, we investigated relationships of bacterial and fungal communities in the rhizosphere and root endosphere of the riparian tree species Populus deltoides, and the influence of soil parameters, environmental properties (host phenotype and aboveground environmental settings), host plant genotype (Simple Sequence Repeat (SSR) markers), season (Spring vs. Fall) and geographic setting (at scales from regional watersheds to local riparian zones) on microbial community structure. Each of the trees sampled displayed unique aspects to its associated community structure with high numbers of Operational Taxonomic Units (OTUs) specific to an individual trees (bacteria >90%, fungi >60%). Over the diverse conditions surveyed only a small number of OTUs were common to all samples within rhizosphere (35 bacterial and 4 fungal) and endosphere (1 bacterial and 1 fungal) microbiomes. As expected, Proteobacteria and Ascomycota were dominant in root communities (>50%) while other higher-level phylogenetic groups (Chytridiomycota, Acidobacteria) displayed greatly reduced abundance in endosphere compared to the rhizosphere. Variance partitioning partially explained differences in microbiome composition between all sampled roots on the basis of seasonal and soil properties (4% to 23%). While most variation remains unattributed, we observed significant differences in the microbiota between watersheds (Tennessee vs. North Carolina) and seasons (Spring vs. Fall). SSR markers clearly delineated two host populations associated with the samples taken in TN vs. NC, but overall host genotypic distances did not have a significant effect on corresponding communities that could be separated from other measured effects.     

Temporal Trends in the Characteristics of Children at Antiretroviral Therapy Initiation in Southern Africa: The IeDEA-SA Collaboration

Background

Since 2005, increasing numbers of children have started antiretroviral therapy (ART) in sub-Saharan Africa and, in recent years, WHO and country treatment guidelines have recommended ART initiation for all infants and very young children, and at higher CD4 thresholds for older children. We examined temporal changes in patient and regimen characteristics at ART start using data from 12 cohorts in 4 countries participating in the IeDEA-SA collaboration.

Methodology/Principal Findings

Data from 30,300 ART-naïve children aged <16 years at ART initiation who started therapy between 2005 and 2010 were analysed. We examined changes in median values for continuous variables using the Cuzick''s test for trend over time. We also examined changes in the proportions of patients with particular disease severity characteristics (expressed as a binary variable e.g. WHO Stage III/IV vs I/II) using logistic regression. Between 2005 and 2010 the number of children starting ART each year increased and median age declined from 63 months (2006) to 56 months (2010). Both the proportion of children <1 year and ≥10 years of age increased from 12 to 19% and 18 to 22% respectively. Children had less severe disease at ART initiation in later years with significant declines in the percentage with severe immunosuppression (81 to 63%), WHO Stage III/IV disease (75 to 62%), severe anemia (12 to 7%) and weight-for-age z-score<−3 (31 to 28%). Similar results were seen when restricting to infants with significant declines in the proportion with severe immunodeficiency (98 to 82%) and Stage III/IV disease (81 to 63%). First-line regimen use followed country guidelines.

Conclusions/Significance

Between 2005 and 2010 increasing numbers of children have initiated ART with a decline in disease severity at start of therapy. However, even in 2010, a substantial number of infants and children started ART with advanced disease. These results highlight the importance of efforts to improve access to HIV diagnostic testing and ART in children.     

Synthesis and structure–activity relationships of phenyl-substituted coumarins with anti-tubercular activity that target FadD32

In an effort to develop new and potent agents for therapy against tuberculosis, a high-throughput screen was performed against Mycobacterium tuberculosis strain H37Rv. Two 6-aryl-5,7-dimethyl-4-phenylcoumarin compounds 1a and 1b were found with modest activity. A series of coumarin derivatives were synthesized to improve potency and to investigate the structure–activity relationship of the series. Among them, compounds 1o and 2d showed improved activity with IC₉₀ of 2 μM and 0.5 μM, respectively. Further optimization provided compound 3b with better physiochemical properties with IC₉₀ 0.4 μM which had activity in a mouse model of infection. The role of the conformation of the 4- and 6-aryl substituents is also described.     

Genetic diversity and karyotype of Pitcairnia azouryi: an endangered species of Bromeliaceae endemic to Atlantic Forest inselbergs

Plant species of various families, such as those of Bromeliaceae, occur on inselbergs where they are subject to geographic isolation and environmental conditions that can lead to genetic erosion. This, in turn, can result in the loss of natural populations due to homozygosis, or changes in ploidy that may lead to reproductive isolation. The genetic diversity of five natural populations of Pitcairnia azouryi was measured using nine microsatellite markers transferred from P. albiflos and P. geyskesii. Chromosome numbers and nuclear DNA content were also evaluated. The results indicated moderate genetic differentiation among populations (F_ST?=?0.188), and significant gene flow (N_m?=?1.073) in four of the five populations. P. azouryi has, predominantly, 2n?=?50 chromosomes and DNA content of 2C?=?1.16 pg, but the tetraploid condition was found (2n?=?100 and 2C?=?2.32 pg) in seedlings of an individual of the most geographically isolated population. The moderate level of genetic structuring observed for P. azouryi seems to be related to its disjoint geographical distribution and the locally aggregated spatial structure of the populations, which are isolated from each other, hindering the inter and intrapopulational gene flow. This interpretation was also evidenced by the mantel test (r?=?0.777, P?<?0.05). The occurrence of diploid individuals with tetraploid seedlings is indicative of events of eupolyploidization, possibly due to the environmental conditions of this geographically isolated population.

     

Species diversity of edaphic mites (Acari: Oribatida) and effects of topography,soil properties and litter gradients on their qualitative and quantitative composition in 64 km<Superscript>2</Superscript> of forest in Amazonia

Small-scale spatial distribution of oribatid mites has been investigated in Amazonia. In addition, medium- and large-scale studies are needed to establish the utility of these mites in detecting natural environmental variability, and to distinguish this variability from anthropogenic impacts. We are expanding the knowledge about oribatid mites in a wet upland forest reserve, and investigate whether a standardized and integrated protocol is an efficient way to assess the effects of environmental variables on their qualitative and quantitative composition on a large spatial scale inside an ecological reserve in Central Amazonia, Brazil. Samples for Berlese-Tullgren extraction were taken in 72 plots of 250 × 6 m distributed over 64 km². In total 3,182 adult individuals, from 82 species and 79 morphospecies were recorded, expanding the number of species known in the reserve from 149 to 254. Galumna, Rostrozetes and Scheloribates were the most speciose genera, and 57 species were rare. Rostrozetes ovulum, Pergalumna passimpuctata and Archegozetes longisetosus were the most abundant species, and the first two were the most frequent. Species number and abundance were not correlated with clay content, slope, pH and litter quantity. However, Principal Coordinate Analysis indicated that as the percentage of clay content, litter quantity and pH changed, the oribatid mite qualitative and quantitative composition also changed. The standardized protocol effectively captured the diversity, as we collected one of the largest registers of oribatid mites’ species for Amazonia. Moreover, biological and ecological data were integrated to capture the effects of environmental variables accounting for their diversity and abundance.