Rapid Detection of Avian Eimeria Species Using Denaturing Gradient Gel Electrophoresis

A denaturing gradient gel electrophoresis (DGGE) assay was developed to rapidly discriminate species of avian Eimeria. Amplification by PCR of the small subunit ribosomal RNA gene (approximately 1,600 nucleotides) with Eimeria genus-specific primers followed by cloning and sequencing allowed us to carry out phylogenetic analyses and identify clone sequences to species level in most cases. Clones were subsequently used to amplify a smaller fragment (approximately 120 nucleotides) suitable for DGGE. The fragments were separated on denaturing gradient gel and bands with unique migration distances were mixed to obtain an identification ladder. The identification ladder and PCR products obtained from DNA extracted from fecal samples from several poultry farms were compared. Applying the DGGE method in this study allowed a rapid differentiation of Eimeria species present in fecal samples collected from poultry farms

Dysbiosis of bifidobacteria and Clostridium cluster XIVa in the cystic fibrosis fecal microbiota

BACKGROUND: Recurrent antimicrobial interventions and disease-related intestinal dysfunction are suspected to contribute to the dysbiosis of the gastrointestinal microbial ecosystem in patients with cystic fibrosis (CF). The present study set out to detect and identify microbial discriminants in the gut microbiota composition that are associated with CF-related intestinal dysbiosis. METHODS: An in-depth description of CF-associated gut dysbiosis was obtained by screening denaturing gradient gel electrophoresis (DGGE) fingerprints for potentially discriminating bacterial species, and quantification by means of real-time PCR analyses using group-specific primers. RESULTS: A total of 8 DGGE band-classes assigned to the genus Bifidobacterium (n=3), and members of Clostridium clusters XIVa (n=3) and IV (n=2), were significantly (p<0.05) underrepresented in samples of patients with CF. Real-time PCR analyses confirmed a significantly lower abundance and temporal stability of bifidobacteria and Clostridium cluster XIVa in the faecal microbiota of patients with CF. CONCLUSION: This study is the first to report specific microbial determinants of dysbiosis in patients with CF

One-step isolation and biochemical characterization of a highlyactive plant PSII monomeric core

We describe a one-step detergent solubilization protocol for isolating a highly active form of Photosystem II (PSII) from Pisum sativum L. Detailed characterization of the preparation showed that the complex was a monomer having no light harvesting proteins attached. This core reaction centre complex had, however, a range of low molecular mass intrinsic proteins as well as the chlorophyll binding proteins CP43 and CP47 and the reaction centre proteins D1 and D2. Of particular note was the presence of a stoichiometric level of PsbW, a low molecular weight protein not present in PSII of cyanobacteria. Despite the high oxygen evolution rate, the core complex did not retain the PsbQ extrinsic protein although there was close to a full complement of PsbO and PsbR and partial level of PsbP. However, reconstitution of PsbP and PsbPQ was possible. The presence of PsbP in absence of LHCII and other chlorophyll a/b binding proteins confirms that LHCII proteins are not a strict requirement for the assembly of this extrinsic polypeptide to the PSII core in contrast with the conclusion of Caffarri et al. (2009)

BACTERIAL COMMUNITY SHIFTS OF A HIGH MOUNTAIN LAKE IN RESPONSE TO VARIABLE SIMULATED CONDITIONS: AVAILABILITY OF NUTRIENTS, LIGHT AND OXYGEN

We studied bacterial population composition shifts by exposing natural water samples to variable simulated environmental conditions. The samples were taken from Lake Jori XIII (2640 m a.s.l), an oligo-to mesotrophic cold freshwater lake, located in the eastern Swiss Alps. The Jori lakes are characterized as remote, unpolluted high mountain lakes with a long period of ice cover and typically low nutrient concentrations. Culture independent techniques (PCR-based analyses) were used for detection and molecular characterization of a large number of bacteria most of which are still uncultivable. Bacterial community shifts over three ecological conditions (nutrients, light and oxygen availability) were detected by using Temporal Temperature gradient Gel Electrophoresis (TTGE) of a PCR-amplified part of the 16S rRNA gene. The bacterial populations responded differently to the variable conditions, as revealed by TTGE pattern shifts during the experiment. Key words: Temporal Temperature gradient Gel Electrophoresis (TTGE), ARB, small subunit ribosomal RNA gene (SSU rRNA gene), alpine freshwater lake JOri XIII, PC

Permanent residents or temporary lodgers: characterizing intracellular bacterial communities in the siphonous green alga Bryopsis

The ecological success of giant celled, siphonous green algae in coastal habitats has repeatedly been linked to endophytic bacteria living within the cytoplasm of the hosts. Yet, very little is known about the relative importance of evolutionary and ecological factors controlling the intracellular bacterial flora of these seaweeds. Using the marine alga Bryopsis (Bryopsidales, Chlorophyta) as a model, we explore the diversity of the intracellular bacterial communities and investigate whether their composition is controlled by ecological and biogeographic factors rather than the evolutionary history of the host. Using a combination of 16S rDNA clone libraries and denaturing gradient gel electrophoresis analyses, we show that Bryopsis harbours a mixture of relatively few but phylogenetically diverse bacterial species. Variation partitioning analyses show a strong impact of local environmental factors on the presence of Rickettsia and Mycoplasma in their association with Bryopsis. The presence of Flavobacteriaceae and Bacteroidetes, on the other hand, reflects a predominant imprint of host evolutionary history, suggesting that these bacteria are more specialized in their association. The results highlight the importance of interpreting the presence of individual bacterial phylotypes in the light of ecological and evolutionary principles such as phylogenetic niche conservatism to understand complex endobiotic communities and the parameters shaping them

The effect of a multispecies probiotic on the composition of the faecal microbiota and bowel habits in chronic obstructive pulmonary disease patients treated with antibiotics

Short-term antibiotic treatment profoundly affects the intestinal microbiota, which may lead to sustained changes in microbiota composition. Probiotics may restore such a disturbance. The objective of the present study was to investigate the effect of a multispecies probiotic on the faecal microbiota during and after antibiotic intake in patients with a history of frequent antibiotic use. In this randomised, placebo-controlled, double-blind study, thirty chronic obstructive pulmonary disease (COPD) patients treated with antibiotics for a respiratory tract infection received 5 g of a multispecies probiotic or placebo twice daily for 2 weeks. Faecal samples were collected at 0, 7, 14 and 63 d. Changes in the composition of the dominant faecal microbiota were determined by PCR-denaturing gradient gel electrophoresis (DGGE). Changes in bacterial subgroups were determined by quantitative PCR and culture. Bowel movements were scored daily according to the Bristol stool form scale. During and after antibiotic treatment, DGGE-based similarity indices (SI) were high ( >/= 84 %) and band richness was relatively low, both remaining stable over time. No difference in SI was observed between patients with and without diarrhoea-like bowel movements. The multispecies probiotic had a modest effect on the bacterial subgroups. Nevertheless, it affected neither the composition of the dominant faecal microbiota nor the occurrence of diarrhoea-like bowel movements. The dominant faecal microbiota was not affected by antibiotics in this COPD population, suggesting an existing imbalance of the microbiota, which may also have contributed to the lack of effect by probiotic intak

Power and limitations of electrophoretic separations in proteomics strategies

Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed

Multiple human herpesvirus-8 infection

In Malawian patients with Kaposi sarcoma (KS) and their relatives, we investigated nucleotide-sequence variation in human herpesvirus-8 (HHV-8) subgenomic DNA, amplified from oral and blood samples by use of polymerase chain reaction. Twenty-four people had amplifiable HHV-8 DNA in >1 sample; 9 (38%) were seropositive for human immunodeficiency virus type 1, 21 (88%) were anti-HHV-8-seropositive, and 7 (29%) had KS. Sequence variation was sought in 3 loci of the HHV-8 genome: the internal repeat domain of open-reading frame (ORF) 73, the KS330 segment of ORF 26, and variable region 1 of ORF K1. Significant intraperson/intersample and intrasample sequence polymorphisms were observed in 14 people (60%). For 3 patients with KS, intraperson genotypic differences, arising from nucleotide sequence variations in ORFs 26 and K1, were found in blood and oral samples. For 2 other patients with KS and for 9 people without KS, intraperson genotypic and subgenotypic differences, originating predominantly from ORF K1, were found in oral samples; for the 2 patients with KS and for 4 individuals without KS, intrasample carriage of distinct ORF K1 sequences also were discernible. Our findings imply HHV-8 superinfection

Two-dimensional gel electrophoresis in proteomics: A tutorial

Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2)

The host genotype affects the bacterial community in the human gastrointestinal tract

The gastrointestinal (GI) tract is one of the most complex ecosystems consisting of microbial and host cells. It is suggested that the host genotype, the physiology of the host and environmental factors affect the composition and function of the bacterial community in the intestine. However, the relative impact of these factors is unknown. In this study, we used a culture-independent approach to analyze the bacterial composition in the GI tract. Denaturing gradient gel electrophoresis (DGGE) profiles of fecal bacterial 16S rDNA amplicons from adult humans with varying degrees of genetic relatedness were compared by determining the similarity indices of the profiles compared. The similarity between fecal DGGE profiles of monozygotic twins were significantly higher than those for unrelated individuals (ts = 2.73, p1-tail = 0.0063, df=21). In addition, a positive relationship (F1, 30 = 8.63, p = 0.0063) between the similarity indices and the genetic relatedness of the hosts was observed. In contrast, fecal DGGE profiles of marital partners, which are living in the same environment and which have comparable feeding habits, showed low similarity which was not significantly different from that of unrelated individuals (ts = 1.03, p1-tail = 0.1561, df=27). Our data indicate that factors related to the host genotype have an important effect on determining the bacterial composition in the GI tract