Nodavirus in farmed Atlantic cod Gadus morhua in Norway

Viral encephalopathy and retinopathy (VER) was diagnosed in 5 to 24 g sized farmed Atlantic cod Gadus morhua kept in sea cages at Parisvatn, Hordaland county, on the west coast of Norway. Moderate mortality (10 to 15%) was observed, along with anorexia and abnormal swimming behaviour, such as looping or spiral swimming and reduced coordination. Nodavirus was detected by 2 different real-time RT-PCR assays, and this was later confirmed by immunohistochemistry. This is the first report of an outbreak of VER in farmed cod in Norway, and the first report that VER affect cod exceeding 5 g in size.     

Preparation of cyclic 2',3'-carbamate derivatives of erythromycin macrolide antibiotics

Tricarbonylation of clarithromycin has been effected in a one-pot reaction with phosgene. The 11,12-diol moiety was closed into a cyclic carbonate, while the dimethylamino alcohol of the desosamine sugar was cyclised with loss of a methyl group to form a cyclic 2',3'-carbamate. The 4' hydroxyl group in clarithromycin was converted into a chloroformate group and subsequently to an allyl carbonate which on Pd-catalysis furnished a novel N-demethylclarithromycin 2',3'-carbamate-11,12-carbonate. Hydrolytic removal of the cladinose sugar and a subsequent oxidation furnished the corresponding ketolide. The 11,12-cyclic carbonate moiety was cleaved by sodium azide to the 10,11-anhydro-9-ketone. 11-N-Arylated cyclic 11,12:2',3'-dicarbamate derivatives were prepared in a copper(I) chloride aided reaction between aryl isocyanates and 10,11-anhydro 9-ketones. The products are novel N-arylated-N'-demethylated 11,12:2',3'-dicarbamate ketolides derived from clarithromycin.     

Automated water analyser computer supported system (AWACSS) Part I: Project objectives, basic technology, immunoassay development, software design and networking

A novel analytical system AWACSS (automated water analyser computer-supported system) based on immunochemical technology has been developed that can measure several organic pollutants at low nanogram per litre level in a single few-minutes analysis without any prior sample pre-concentration nor pre-treatment steps. Having in mind actual needs of water-sector managers related to the implementation of the Drinking Water Directive (DWD) (98/83/EC, 1998) and Water Framework Directive WFD (2000/60/EC, 2000), drinking, ground, surface, and waste waters were major media used for the evaluation of the system performance. The instrument was equipped with remote control and surveillance facilities. The system's software allows for the internet-based networking between the measurement and control stations, global management, trend analysis, and early-warning applications. The experience of water laboratories has been utilised at the design of the instrument's hardware and software in order to make the system rugged and user-friendly. Several market surveys were conducted during the project to assess the applicability of the final system. A web-based AWACSS database was created for automated evaluation and storage of the obtained data in a format compatible with major databases of environmental organic pollutants in Europe. This first part article gives the reader an overview of the aims and scope of the AWACSS project as well as details about basic technology, immunoassays, software, and networking developed and utilised within the research project. The second part article reports on the system performance, first real sample measurements, and an international collaborative trial (inter-laboratory tests) to compare the biosensor with conventional anayltical methods.     

Electroneutral sodium absorption and electrogenic anion secretion across murine small intestine are regulated in parallel

Electrolyte transport processes of small intestinal epithelia maintain a balance between hydration of the luminal contents and systemic fluid homeostasis. Under basal conditions, electroneutral Na(+) absorption mediated by Na(+)/H(+) exchanger 3 (NHE3) predominates; under stimulated conditions, increased anion secretion mediated by CFTR occurs concurrently with inhibition of Na(+) absorption. Homeostatic adjustments to diseases that chronically affect the activity of one transporter (e.g., cystic fibrosis) may include adaptations in the opposing transport process to prevent enterosystemic fluid imbalance. To test this hypothesis, we measured electrogenic anion secretion (indexed by the short-circuit current) across NHE3-null [NHE3(-)] murine small intestine and electroneutral Na(+) absorption (by radioisotopic flux analysis) across small intestine of mice with gene-targeted disruptions of the anion secretory pathway, i.e., CFTR-null [CFTR(-)] or Na(+)-K(+)-2Cl(-) cotransporter-null [NKCC1(-)]. Protein expression of NHE3 and CFTR in the intestinal epithelia was measured by immunoblotting. In NHE3(-), compared with wild-type small intestine, maximal and bumetanide-sensitive anion secretion following cAMP stimulation was significantly reduced, and there was a corresponding decrease in CFTR protein expression. In CFTR(-) and NKCC1(-) intestine, Na(+) absorption was significantly reduced compared with wild-type. NHE3 protein expression was decreased in the CFTR(-) intestine but was unchanged in the NKCC1(-) intestine, indicating that factors independent of expression also downregulate NHE3 activity. Together, these data support the concept that absorptive and secretory processes determining NaCl and water movement across the intestinal epithelium are regulated in parallel to maintain balance between the systemic fluid volume and hydration of the luminal contents.     

Epitope analyses of pneumococcal surface protein A: a combination of two monoclonal antibodies detects 94% of clinical isolates

Immunisation of BALB/c mice with seven heat-treated Norwegian clinical isolates of Streptococcus pneumoniae of different serotypes elicited mainly monoclonal antibodies (mAbs) to pneumococcal surface protein A (PspA). It was remarkable that the fusions resulted only in a few mAbs directed against other protein antigens. Dot blot analysis with 16 mAbs using clinical isolates representing 23 different capsular types and the uncapsulated reference strain R36A showed that some of the mAbs bound to PspA epitopes expressed by a low number of strains whereas others bound to broadly distributed epitopes. On the basis of their reactivities, seven of these mAbs could be divided into two groups recognising different subsets of pneumococci. The three mAbs in the narrow reacting group bound to epitopes found in 21-25% of the strains whereas the four mAbs in the broad reacting group detected more than 57% of the analysed strains. The epitopes for these seven antibodies were surface exposed on live exponential phase grown pneumococci as shown by flow cytometry. The finding that a combination of mAb 180,C-1 (IgG2a) from the first group and mAb 170,E-11 (IgG2a) from the second group detected 94% of the examined strains is interesting because PspA has been reported by others to be a serological highly variable protein.     

A new genus of Platycopioida (Copepoda) from a marine cave on Bermuda

Nanocopia minuta was collected from an inland marine cave on Bermuda. This new genus is reminiscent of Platycopia in its cephalic appendages and in the 5th legs of the male. The 1st leg has a 3-segmented exopod and a 1-segmented endopod. The other legs show reductions with no armature along the inner margin of the exopods and with 2-segmented endopods in the female. Only 2nd and 3rd legs bear two outer spines on the first exopodal segment.Owing to similarities in sexual characters, mouthparts, and modifications of the swimming legs, Nanocopia and Platycopia are considered more closely related to each other than either is to Antrisocopia.Platycopioida now contains three genera of which two are found only in Roadside Cave. The order has retained several primitive characters and seems to have separated early from the gymnoplean stem.     

Evaluation of Tumor Cell Proliferation by Ki-67 Expression and Mitotic Count in Lymph Node Metastases from Breast Cancer

Few studies have addressed the risk of recurrence by assessing proliferation markers in lymph node metastasis from breast cancer. Here, we aimed to examine Ki-67 expression and mitotic count in lymph nodes in comparison with primary tumors. A cohort of node positive breast cancer (n = 168) was studied as a part of the prospective Norwegian Breast Cancer Screening Program (1996–2009). The percentage of Ki-67 positivity was counted per 500 tumor cells in hot-spot areas (x630). Mitotic count was conducted in the most cellular and mitotic active areas in 10 high power fields (x400). Our results showed that Ki-67 and mitotic count were significantly correlated between primary tumor and lymph nodes (Spearman`s correlation 0. 56 and 0.46, respectively) and were associated with most of the histologic features of the primary tumor. Univariate survival analysis (log-rank test) showed that high Ki-67 and mitotic count in the primary tumor and lymph node metastasis significantly predicted risk of recurrence. In multivariate analysis, mitotic count in the lymph node metastasis was an independent predictor of tumor recurrence. In conclusion, proliferation markers in lymph node metastases significantly predicted disease free survival in node positive breast cancer.     

A Multicentre Hospital Outbreak in Sweden Caused by Introduction of a vanB2 Transposon into a Stably Maintained pRUM-Plasmid in an Enterococcus faecium ST192 Clone

The clonal dissemination of VanB-type vancomycin-resistant Enterococcus faecium (VREfm) strains in three Swedish hospitals between 2007 and 2011 prompted further analysis to reveal the possible origin and molecular characteristics of the outbreak strain. A representative subset of VREfm isolates (n = 18) and vancomycin-susceptible E. faecium (VSEfm, n = 2) reflecting the spread in time and location was approached by an array of methods including: selective whole genome sequencing (WGS; n = 3), multi locus sequence typing (MLST), antimicrobial susceptibility testing, virulence gene profiling, identification of mobile genetic elements conferring glycopeptide resistance and their ability to support glycopeptide resistance transfer. In addition, a single VREfm strain with an unrelated PFGE pattern collected prior to the outbreak was examined by WGS. MLST revealed a predominance of ST192, belonging to a hospital adapted high-risk lineage harbouring several known virulence determinants (n≥10). The VREfm outbreak strain was resistant to ampicillin, gentamicin, ciprofloxacin and vancomycin, and susceptible to teicoplanin. Consistently, a vanB2-subtype as part of Tn1549/Tn5382 with a unique genetic signature was identified in the VREfm outbreak strains. Moreover, Southern blot hybridisation analyses of PFGE separated S1 nuclease-restricted total DNAs and filter mating experiments showed that vanB2-Tn1549/Tn5382 was located in a 70-kb sized rep _17/pRUM plasmid readily transferable between E. faecium. This plasmid contained an axe-txe toxin-antitoxin module associated with stable maintenance. The two clonally related VSEfm harboured a 40 kb rep _17/pRUM plasmid absent of the 30 kb vanB2-Tn1549/Tn5382 gene complex. Otherwise, these two isolates were similar to the VREfm outbreak strain in virulence- and resistance profile. In conclusion, our observations support that the origin of the multicentre outbreak was caused by an introduction of vanB2-Tn1549/Tn5382 into a rep _17/pRUM plasmid harboured in a pre-existing high-risk E. faecium ST192 clone. The subsequent dissemination of VREfm to other centres was primarily caused by clonal spread rather than plasmid transfer to pre-existing high-risk clones.