An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.     

Models of foot-and-mouth disease

During the 2001 foot-and-mouth disease outbreak in the UK, three very different models were used in an attempt to predict the disease dynamics and inform control measures. This was one of the first times that models had been used during an epidemic to support the decision-making process. It is probable that models will play a pivotal role in any future livestock epidemics, and it is therefore important that decision makers, veterinarians and farmers understand the uses and limitations of models. This review describes the utility of models in general before focusing on the three foot-and-mouth disease models used in 2001. Finally, the future of modelling is discussed, analysing the advances needed if models are to be successfully applied during any subsequent epidemics.     

GObar: A Gene Ontology based analysis and visualization tool for gene sets

Background  

Microarray experiments, as well as other genomic analyses, often result in large gene sets containing up to several hundred genes. The biological significance of such sets of genes is, usually, not readily apparent.     

Appropriate models for the management of infectious diseases

BackgroundMathematical models have become invaluable management tools for epidemiologists, both shedding light on the mechanisms underlying observed dynamics as well as making quantitative predictions on the effectiveness of different control measures. Here, we explain how substantial biases are introduced by two important, yet largely ignored, assumptions at the core of the vast majority of such models.ConclusionThis work aims to highlight that, when developing models for public health use, we need to pay careful attention to the intrinsic assumptions embedded within classical frameworks.     

Diversity and evolutionary history of plastids and their hosts

By synthesizing data from individual gene phylogenies, large concatenated gene trees, and other kinds of molecular, morphological, and biochemical markers, we begin to see the broad outlines of a global phylogenetic tree of eukaryotes. This tree is apparently composed of five large assemblages, or "supergroups." Plants and algae, or more generally eukaryotes with plastids (the photosynthetic organelle of plants and algae and their nonphotosynthetic derivatives) are scattered among four of the five supergroups. This is because plastids have had a complex evolutionary history involving several endosymbiotic events that have led to their transmission from one group to another. Here, the history of the plastid and of its various hosts is reviewed with particular attention to the number and nature of the endosymbiotic events that led to the current distribution of plastids. There is accumulating evidence to support a single primary origin of plastids from a cyanobacterium (with one intriguing possible exception in the little-studied amoeba Paulinella), followed by the diversification of glaucophytes, red and green algae, with plants evolving from green algae. Following this, some of these algae were themselves involved in secondary endosymbiotic events. The best current evidence indicates that two independent secondary endosymbioses involving green algae gave rise to euglenids and chlorarachniophytes, whereas a single endosymbiosis with a red algae gave rise to the chromalveolates, a diverse group including cryptomonads, haptophytes, heterokonts, and alveolates. Dinoflagellates (alveolates) have since taken up other algae in serial secondary and tertiary endosymbioses, raising a number of controversies over the origin of their plastids, and by extension, the recently discovered cryptic plastid of the closely related apicomplexan parasites.     

Evidence from SSU rRNA phylogeny that Octomitus is a sister lineage to Giardia

Octomitus intestinalis is a diplomonad flagellate inhabiting the digestive tract of rodents and amphibians. Octomitus is of evolutionary interest because, based on ultrastructural characteristics, it is thought to be closely related to the morphologically derived genus Giardia, and together they have been proposed to make up the Giardiinae. In molecular trees of diplomonads, Giardia is the deepest branching lineage, so identifying a sister group to Giardia that is less derived would be informative. Octomitus is a logical candidate for this position, but unfortunately there are no molecular data from it, and it is not available in culture. To determine the position of Octomitus, and specifically test whether it is more closely related to Giardia than other diplomonads, we have isolated it directly from the caecum of wild mice and characterized its small subunit ribosomal RNA (SSU rRNA) gene. Phylogenetic analysis showed Octomitus to be the sister to Giardia with strong support, together occupying one side of the deepest split in the diplomonad tree.     

Diterpene resin acids in conifers

Diterpene resin acids are a significant component of conifer oleoresin, which is a viscous mixture of terpenoids present constitutively or inducibly upon herbivore or pathogen attack and comprises one form of chemical resistance to such attacks. This review focuses on the recent discoveries in the chemistry, biosynthesis, molecular biology, regulation, and biology of these compounds in conifers.     

Relative rates of evolution among the three genetic compartments of the red alga Porphyra differ from those of green plants and do not correlate with genome architecture

In photosynthetic eukaryotes, relative silent-site nucleotide substitution rates (which can be used to approximate relative mutation rates) among mitochondrial, plastid, and nuclear genomes (mtDNAs, ptDNAs, and nucDNAs) are estimated to be 1:3:10 respectively for seed plants and roughly equal for green algae. These estimates correlate with certain genome characteristics, such as size and coding density, and have therefore been taken to support a relationship between mutation rate and genome architecture. Plants and green algae, however, represent a small fraction of the major eukaryotic plastid-bearing lineages. Here, we investigate relative rates of mutation within the model red algal genus Porphyra. In contrast to plants, we find that the levels of silent-site divergence between the Porphyra purpurea and Porphyra umbilicalis mtDNAs are three times that of their ptDNAs and five times that of their nucDNAs. Moreover, relative mutation rates do not correlate with genome architecture: despite an estimated three-fold difference in their mutation rate, the mitochondrial and plastid genome coding densities are equivalent - an observation that extends to organisms with secondary red algal plastids. These findings are supported by within-species silent-site polymorphism data from P. purpurea.     

Evaluating the Ribosomal Internal Transcribed Spacer (ITS) as a Candidate Dinoflagellate Barcode Marker

Background

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Methodology/Principal Findings

The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.

Conclusions/Significance

The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.