Direct real-time PCR quantification of Campylobacter jejuni in chicken fecal and cecal samples by integrated cell concentration and DNA purification

Campylobacter jejuni is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of C. jejuni in poultry is the cecum, with an estimated content of 6 to 8 log10 CFU/g. If a flock is infected with C. jejuni, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for C. jejuni obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5'-nuclease PCR. The assay was evaluated both by spiking the samples with C. jejuni and through the detection of C. jejuni in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log10 were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were Campylobacter positive, whereas 12 were negative. Two of the flocks contained Campylobacter species other than C. jejuni. There was a large difference in the C. jejuni content, ranging from 4 to 8 log10 CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of C. jejuni to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.     

The Frequency of Silencing in Arabidopsis Thaliana Varies Highly Between Progeny of Siblings and can be Influenced by Environmental Factors

In a collection of 111 transgenic Arabidopsis thaliana lines, silencing of the nptII gene was observed in 62 (56%) of the lines and three distinct nptII-silencing phenotypes were identified. Two T-DNA constructs were used, which differed in distance and orientation of the marker gene relative to the border sequences. Comparison of the sets of lines generated with each vector, indicate that the T-DNA construct configuration influence the incidence of lines displaying silencing, as well as the distribution of silencing phenotypes. Twenty lines were investigated more thoroughly. The frequency of silencing varied between siblings in 19 lines, including three lines containing a single T-DNA copy. The last line showed 100% silencing. The gus gene present in both constructs could be expressed in the presence of a silenced nptII gene. Investigation of methylation at a single site in the pnos promoter revealed partial methylation in multi-copy lines, but no methylation in single-copy lines. For 16 lines, the overall frequencies of silencing differed significantly between control plants and plants exposed to temperature stress; in 11 of these lines at the 0.1% level. In several cases, the frequency of silencing in progeny of stress-treated plants was higher than for the control group, while other lines showed higher frequencies of kanamycin-resistant progeny for the stress-treated sibling plants.     

Analysis of expressed sequence tags from the marine microalga Pseudochattonella farcimen (Dictyochophyceae)

Pseudochattonella farcimen (Eikrem, Edvardsen, et Throndsen) is a unicellular alga belonging to the Dictyochophyceae (Heterokonta). It forms recurring blooms in Scandinavian coastal waters, and has been associated to fish mortality. Here we report the sequencing and analysis of 10,368 expressed sequence tags (ESTs) corresponding to 8,149 unique gene models from this species. Compared to EST libraries from other heterokonts, P. farcimen contains a high number of genes with functions related to cell communication and signaling. We found several genes encoding proteins related to fatty acid metabolism, including eight fatty acid desaturases and two phospholipase A2 genes. Three desaturases are highly similar to Δ4-desaturases from haptophytes. P. farcimen also possesses three putative polyketide synthases (PKSs), belonging to two different families. Some of these genes may have been acquired via horizontal gene transfer by a common ancestor of brown algae and dictyochophytes, together with genes involved in mannitol metabolism, which are also present in P. farcimen. Our findings may explain the unusual fatty acid profile previously observed in P. farcimen, and are discussed from an evolutionary perspective and in relation to the ichthyotoxicity of this alga.     

Switching on the light: using metagenomic shotgun sequencing to characterize the intestinal microbiome of Atlantic cod

Atlantic cod (Gadus morhua) is an ecologically important species with a wide-spread distribution in the North Atlantic Ocean, yet little is known about the diversity of its intestinal microbiome in its natural habitat. No geographical differentiation in this microbiome was observed based on 16S rRNA amplicon analyses, yet such finding may result from an inherent lack of power of this method to resolve fine-scaled biological complexity. Here, we use metagenomic shotgun sequencing to investigate the intestinal microbiome of 19 adult Atlantic cod individuals from two coastal populations in Norway–located 470 km apart. Resolving the species community to unprecedented resolution, we identify two abundant species, Photobacterium iliopiscarium and Photobacterium kishitanii, which comprise over 50% of the classified reads. Interestingly, the intestinal P. kishitanii strains have functionally intact lux genes, and its high abundance suggests that fish intestines form an important part of its ecological niche. These observations support a hypothesis that bioluminescence plays an ecological role in the marine food web. Despite our improved taxonomical resolution, we identify no geographical differences in bacterial community structure, indicating that the intestinal microbiome of these coastal cod is colonized by a limited number of closely related bacterial species with a broad geographical distribution.     

Long‐read sequence capture of the haemoglobin gene clusters across codfish species

Combining high‐throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short‐read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long‐read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom‐made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage‐specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long‐read capture as a versatile approach for comparative genomic studies by generation of a cross‐species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes.     

Alginate stability during high salt preservation of Ascophyllum nodosum

Formaldehyde is usually added to brown algae to avoid microbial growth during storage and to fix polyphenols in the algae before alginate extraction. Since formaldehyde is toxic, allergenic and possibly carcinogenic, dry salting of Ascophyllum nodosum was tested as an alternative. The seaweeds, harvested at locations with a salinity of about 30‰ from late autumn to early spring, were stored at 22±2 °C under compost-like conditions. Untreated samples of seaweed lost their quality as a raw material for alginate production within 14 days. Salted (20–22%) as well as formaldehyde treated seaweed was preserved for at least 46 days. Due to the reduced water activity and oxygen saturation in the dry salted seaweed, microbial growth and brown colouring reactions were suppressed. Economic factors must also be taken into account before large-scale applications are considered. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct determination of alginate content in brown algae by near infra-red (NIR) spectroscopy

The methods used to quantify total alginate in brown algal tissue are time-consuming and may also be misleading, so faster and simpler methods for measuring alginate content would be beneficial in a variety of applications. This study reports on the use of near infra-red (NIR) analysis to monitor the alginate content of Laminaria hyperborea stipe during biodegradation. NIR reflectance spectra were recorded for 78 different freeze-dried samples of its stipe. The samples were collected during several biological degradation experiments and the total alginate content varied from 2.2 to 40.8% Na-alginate (w/w), determined by established methods based on ion exchange. Data analysis was performed using multivariate calibration methods in order to relate the spectral data to the alginate content. PLS2 analysis revealed some dependence on material type, probably reflecting differences in polyphenol content. In the end, a PLS1 model with 9 components was selected. The calculated model was validated both with internal data and with an external test set. Internal full cross validation explained 96.6% of the variance in alginate content. The external validation showed that the PLS1 model was able to predict the alginate concentration with a root mean square prediction accuracy of 2.1%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Complex Evolutionary Patterns of tRNAUAALeu Group I Introns in Cyanobacterial Radiation

Based on the findings that plastids and cyanobacteria have similar group I introns inserted into tRNAUAALeu genes, these introns have been suggested to be immobile and of ancient origin. In contrast, recent evidence suggests lateral transfer of cyanobacterial group I introns located in tRNAUAALeu genes. In light of these new findings, we have readdressed the evolution and lateral transfer of tRNAUAALeu group I introns in cyanobacteral radiation. We determined the presence of introns in 38 different strains, representing the major cyanobacterial lineages, and characterized the introns in 22 of the strains. Notably, two of these strains have two tRNAUAALeu genes, with each of these genes interrupted by introns, while three of the strains have both interrupted and uninterrupted genes. Two evolutionary distinct clusters of tRNA genes, with the genes interrupted by introns belonging to two distinct intron clusters, were identified. We also compared 16S rDNA and intron evolution for both closely and distantly related strains. The distribution of the introns in the clustered groups, as defined from 16S rDNA analysis, indicates relatively recent gain and/or loss of the introns in some of these lineages. The comparative analysis also suggests differences in the phylogenetic trees for 16S rDNA and the tRNAUAALeu group I introns. Taken together, our results show that the evolution of the intron is considerably more complex than previous studies found to be the case. We discuss, based on our results, evolutionary models involving lateral intron transfer and models involving differential loss of the intron.