Diseases of farmed Atlantic salmon Salmo salar associated with infections by the microsporidian Paranucleospora theridion.

The microsporidian Paranucleospora theridion was discovered in Atlantic salmon Salmo salar suffering from proliferative gill disease in a marine farm in western Norway in 2008. The parasite develops in cells of the reticuloendothelial system, cells important for normal immune function. The aim of this study was to see if P. theridion could play a part in some of the diseases with unclear causes in salmon production in Norway, i.e. proliferative gill disease (PGI), pancreas disease (PD), heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS). P. theridion was present in all areas with salmon farming in Norway, but high prevalence and densities of the parasite in salmon and salmon lice were only seen in southern Norway. This region is also the main area for PGI and PD in Norway. Quantification of pathogens associated with PGI, PD, HSMI and CMS diagnoses showed that P. theridion levels are high in southern Norway, and may therefore play a role in susceptibility and disease development. However, among the different diagnoses, fish with PGI are particularly heavily infected with P. theridion. Therefore, P. theridion appears as a possible primary agent in cases with high mortality in connection with PGI in western Norway.

Design principles for simulation games for learning clinical reasoning: A design-based research approach.

BACKGROUND: Nurses sometimes lack the competence needed for recognising deterioration in patient conditions and this is often due to poor clinical reasoning. There is a need to develop new possibilities for learning this crucial competence area. In addition, educators need to be future oriented; they need to be able to design and adopt new pedagogical innovations. The purpose of the study is to describe the development process and to generate principles for the design of nursing simulation games. METHOD: A design-based research methodology is applied in this study. Iterative cycles of analysis, design, development, testing and refinement were conducted via collaboration among researchers, educators, students, and game designers. RESULTS: The study facilitated the generation of reusable design principles for simulation games to guide future designers when designing and developing simulation games for learning clinical reasoning. CONCLUSION: This study makes a major contribution to research on simulation game development in the field of nursing education. The results of this study provide important insights into the significance of involving nurse educators in the design and development process of educational simulation games for the purpose of nursing education.

A large sample of finnish diabetic sib-pairs reveals no evidence for a non-insulin-dependent diabetes mellitus susceptibility locus at 2qter.

In the first reported positive result from a genome scan for non-insulin-dependent diabetes mellitus (NIDDM), Hanis et al. found significant evidence of linkage for NIDDM on chromosome 2q37 and named the putative disease locus NIDDM1 (Hanis et al. 1996. Nat. Genet. 13:161-166). Their total sample was comprised of 440 Mexican-American affected sib-pairs from 246 sibships. The strongest evidence for linkage was at marker D2S125 and best estimates of lambdas (risk to siblings of probands/population prevalence) using this marker were 1.37 under an additive model and 1.36 under a multiplicative model. We examined this chromosomal region using linkage analysis in a Finnish sample comprised of 709 affected sib-pairs from 472 sibships. We excluded this region in our sample (multipoint logarithm of odds score /= 1.37. We discuss possible reasons why linkage to 2q37 was not found and conclude that this region is unlikely to be playing a major role in NIDDM susceptibility in the Finnish Caucasian population.

Detection of minimal residual disease using fluorescence DNA in situ hybridization: a follow-up study in leukemia and lymphoma patients.

We used fluorescence DNA in situ hybridization (FISH) to detect chromosomal abnormalities as an indicator of minimal residual disease in follow-up samples from the bone marrow (BM), or peripheral blood, of 25 patients with leukemia, lymphoma and myelodysplastic syndromes. Trisomies were detected by interphase FISH with repeat-sequence probes (RSP) or by using metaphase FISH with whole-chromosome paint probes (WCP). Specific translocations were detected using WCP probes. Translocations were observed using metaphase FISH in two patients in uncertain or complete remission (CR), who both later suffered relapse. Five patients with no abnormal cells remained in CR. Four patients with trisomies detected during CR suffered relapse; metaphase FISH detected the trisomy in 0.17-16% of metaphase cells. Five patients for whom the trisomy occurred in 0.034% of cells remained in CR. Trisomic nuclei were observed in 0.27-2.3% of interphase cells, by means of RSPs, in four patients who later suffered relapse. Five patients with trisomic nuclei in 0.061% remained in CR. When two probes were used simultaneously in a sample from one patient, 1% of the residual cells were abnormal. The patient later suffered relapse. In one patient with anaplastic large cell lymphoma, CD30-positive interphase cells were shown to have trisomic chromosome 7 by immunophenotyping and FISH. Our results suggest that metaphase FISH using WCP probes is a sensitive and specific method for detecting minimal residual disease especially in patients with translocations.

Mapping genes for NIDDM. Design of the Finland-United States Investigation of NIDDM Genetics (FUSION) Study.

OBJECTIVE: To map and identify susceptibility genes for NIDDM and for the intermediate quantitative traits associated with NIDDM. RESEARCH DESIGN AND METHODS: We describe the methodology and sample of the Finland-United States Investigation of NIDDM Genetics (FUSION) study. The whole genome search approach is being applied in studies of several different ethnic groups to locate susceptibility genes for NIDDM. Detailed description of the study materials and designs of such studies are important, particularly when comparing the findings in these studies and when combining different data sets. RESULTS: Using a careful selection strategy, we have ascertained 495 families with confirmed NIDDM in at least two siblings and no history of IDDM among the first-degree relatives. These families were chosen from more than 22,000 NIDDM patients, representative of patients with NIDDM in the Finnish population. In a subset of families, a spouse and offspring were sampled, and they participated in a frequently sampled intravenous glucose tolerance test (FSIGT) analyzed with the Minimal Model. An FSIGT was completed successfully for at least two nondiabetic offspring in 156 families with a confirmed nondiabetic spouse and no history of IDDM in first-degree relatives. CONCLUSIONS: Our work demonstrates the feasibility of collecting a large number of affected sib-pair families with NIDDM to provide data that will enable a whole genome search approach, including linkage analysis.

Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning.

The Applied Biosystems PRISM fluorescence-based genotyping system as well as the Invitrogen TA Cloning vector system are influenced by the tendency of Taq DNA polymerase to add an adenine nucleotide to the 3' end of PCR products after extension. Incomplete addition of adenine to a majority of PCR product strands creates problems in allele-calling during genotyping and potentially diminishes the cloning efficiency of such products. Experiments reported here show that certain terminal nucleotides can either inhibit or enhance adenine addition by Taq and that PCR primer design can be used to modulate this activity. The methods we propose can substantially improve allele-calling for problematic microsatellite markers when using GENOTYPER software.

Molecular characterisation of the nucleocapsid protein gene, glycoprotein gene and gene junctions of rhabdovirus 903/87, a novel fish pathogenic rhabdovirus.

The sequences of the nucleocapsid and glycoprotein genes and the gene junctions of the fish pathogenic rhabdovirus 903/87 were determined from cDNA and PCR clones. The mRNA of the nucleocapsid is most likely 1492 nucleotides long and encodes a protein of 426 amino acids, whereas the mRNA of the glycoprotein is likely to be 1682 nucleotides long and the protein 517 amino acids. When the nucleocapsid and glycoprotein genes of virus 903/87 were compared at amino acid level with other rhabdoviruses they showed the highest homology with the Vesiculovirus genus. By sequencing the junctions between the N, P, M, G and L genes it was determined that transcription start and stop codons were conserved between virus 903/87 and the vesiculoviruses. Virus 903/87 has no open reading frame coding for a non-virion gene between the glycoprotein and the polymerase gene. Phylogenetic studies based on rhabdovirus nucleocapsid and glycoprotein genes suggested that virus 903/87 is related to viruses in the Vesiculovirus genus.

Analysis of genotype and phenotype on the same interphase or mitotic cell. A manual of MAC (morphology antibody chromosomes) methodology.

The purpose of this paper is to serve as a MAC (Morphology Antibody Chromosome) manual describing combined methodologies that allow simultaneous and/or sequential analysis of cell morphology, immunophenotype, and banded chromosomes and/or in situ hybridization signals. The MAC techniques used at the Department of Medical Genetics of the University of Helsinki, Finland, are described and modifications or related techniques reported by other authors are discussed. A list of references concerning applications is also given.

Cell lineage involvement in four patients with myelodysplastic syndrome and t(1;7) or trisomy 8 studied by simultaneous immunophenotyping and fluorescence in situ hybridization.

Four patients with myelodysplastic syndrome (MDS), one with t(1;7) and three with trisomy 8, were studied by immunophenotyping and fluorescence in situ hybridization (FISH) to assess cell lineage involvement. The t(1;7) was detected using a biotin-labeled chromosome 1 centromere-specific DNA probe. This aberration was present in CD34-positive stem cells, the erythroid cell lineage (GPA+), and the granulocytic/monocytic (CD13+ and CD64+) cell lineages. We were not able to demonstrate the abnormality in the lymphoid cell lineages. In the patients with trisomy 8, the aberration was detected with chromosome 8 centromere-specific DNA probe or by chromosome in situ suppression hybridization (CISS) with a chromosome 8-specific library probe. The trisomy was detected in stem cells, erythroid precursor cells, megakaryocytes, and granulocytes/monocytes. In these MDS patients, the chromosome aberrations appear to occur only in cells of myeloid lineage.

Morphogenesis of salmonid gill poxvirus associated with proliferative gill disease in farmed Atlantic salmon (Salmo salar) in Norway.

Proliferative gill disease (PGD) is an emerging problem in Norwegian culture of Atlantic salmon (Salmo salar). Parasites (Ichthyobodo spp.) and bacteria (Flexibacter/Flavobacterium) may cause PGD, but for most cases of PGD in farmed salmon in Norway, no specific pathogen has been identified as the causative agent. However, Neoparamoeba sp. and several bacteria and viruses have been associated with this disease. In the spring of 2006, a new poxvirus, salmon gill poxvirus (SGPV), was discovered on the gills of salmon suffering from PGD in fresh water in northern Norway. Later the same year, this virus was also found on gills of salmon at two marine sites in western Norway. All farms suffered high losses associated with the presence of this virus. In this study, we describe the entry and morphogenesis of the SGP virus in epithelial gill cells from Atlantic salmon. Intracellular mature virions (IMVs) are the only infective particles that seem to be produced. These are spread by cell lysis and by "budding" of virus packages, containing more that 100 IMVs, from the apical surface of infected cells. Entry of the IMVs appears to occur by attachment to microridges on the cell surface and fusion of the viral and cell membranes, delivering the cores into the cytoplasm. The morphogenesis starts with the emergence of crescents in viroplasm foci in perinuclear areas of infected cells. These crescents consist of two tightly apposed unit membranes (each 5 nm thick) that seem to be derived from membranes of the endoplasmic reticulum. The crescents develop into spheres, immature virions (IVs), that are 350 nm in diameter and surrounded by two unit membranes. The maturation of the IVs occurs by condensation of the core material and a change from spherical to boat-shaped particles, intracellular mature virions (IMVs), that are about 300 nm long. Hence, the IMVs from the SGP virus have a different morphology compared to other vertebrate poxviruses that are members of the subfamily Chordopoxvirinae, and they are more similar to members of subfamily Entomopoxvirinae, genus Alphaentomopoxvirus. However, it is premature to make a taxonomic assignment until the genome of the SGP virus has been sequenced, but morphogenesis clearly shows that this virus is a member of family Poxviridae.

New clade of betanodaviruses detected in wild and farmed cod (Gadus morhua) in Norway.

Betanodaviruses have been isolated and detected in both farmed and wild fish species worldwide. They are classified in five clusters, and all are connected to mortalities in farmed fish. The clusters do not represent specific geographical areas or host species, but one cluster, barfin flounder nervous necrosis virus (BFNNV), is mainly associated with cold water fish species. This study presents the first species-specific clade within the BFNNV cluster. This clade consists of six isolates from wild and farmed Atlantic cod in Norway and is genetically distinct from other betanodaviruses in the North Atlantic. Screening of farmed and wild cod in Norway shows that betanodaviruses are present in wild fish on the west coast of Norway, including migratory cod, but so far we have not detected any betanodavirus-positive wild cod in northern Norway. The presence of significant amounts of betanodaviruses in wild cod represents a serious challenge for the management of viral nervous necrosis in farmed cod in Norway. Betanodavirus-positive farmed cod were present both in western and northern Norway. Mortalities in three cod farms were suspected to be caused by betanodaviruses; however, in two of these, other pathogens may have been responsible for or strongly contributed to the mortalities.

Metaphase fluorescence in situ hybridization (FISH) in the follow-up of 60 patients with haemopoietic malignancies.

Metaphase DNA fluorescence in situ hybridization (metaphase-FISH) was performed on follow-up samples from 60 patients suffering from haemopoietic malignancies (acute and chronic myeloid leukaemia, acute lymphoblastic leukaemia, non-Hodgkin's lymphoma and myelodysplastic syndrome). All patients had clonal chromosomal trisomies or translocations at diagnosis, and were treated by bone marrow transplantation (BMT), chemotherapy (CT) or interferon-alpha therapy. Metaphase-FISH was performed during therapy-induced complete haematological remission (CR) (BMT and CT patients) using biotin-labelled whole chromosome paint probes. 28% of all patients in CR were shown by FISH to have abnormal metaphase cells, and 62% of this group suffered a clinical relapse. Of those with negative FISH results (72%), 12% relapsed. In three CML patients treated with BMT a small population of t(9;22)-positive cells was demonstrated. These cells disappeared during follow-up without causing a relapse. One ALL patient had abnormal cells a short time after start of therapy but was also later found FISH-negative. Furthermore, we demonstrated that metaphase-FISH is a suitable method for quantifying the proportion of abnormal cells in CML patients during interferon-alpha therapy. Metaphase-FISH was also employed to detect a local relapse in an ALL patient. Thus, metaphase-FISH was found reliable and sensitive for detection of minimal residual disease in patients with haemopoietic malignancies.

Postgraduate medical training for deck officers.

347 Finnish deck officers completed the questionnaire on medical training, knowledge and skills. The following conclusions could be drawn: a. Medical training must be based on generally accepted standards, both nationally and internationally. b. More practical exercises should be included in the training. c. Refresher medical training clearly increases knowledge and skills but it also gives the possibility to train, maintain and repeat practical routines. d. Evaluation of the skills should be a part of qualification. e. Good medical knowledge on board ship needs radio-medical services and vice versa.

Simultaneous detection of high-resolution R-banding and fluorescence in situ hybridization signals after fluorouracil-induced cellular synchronization.

A method for simultaneous detection of fluorescence in situ hybridization of DNA probes and high resolution fluorescent R banding is described. Human lymphocytes were stimulated with phytohemagglutinin and synchronized using a fluorouracil block followed by exposure to bromodeoxyuridine and Hoechst 33258 prior to harvest. Metaphase preparations were treated with Hoechst 33258 and exposed to UV light. Thereafter they were incubated in sodium phosphate buffer and dried prior to in situ hybridization with a biotin-labelled centromere-specific alpha-satellite DNA probe for chromosome 1 (pUCl.77) and two digoxigenin-labelled probes, i.e., a PCR-generated chromosome 8-specific alphoid probe (#8) and a cosmid probe for FLT4 gene on 5q33-qter (class III receptor tyrosine kinase). Hybridization signals were detected by an indirect immunofluorescence method using fluorescein isothiocyanate. The chromosomes were counterstained with propidium iodide and 4',6-diamidino-2-phenylindole dihydrochloride. This simple method allows unambiguous chromosome band identification simultaneously with detection of the hybridized probes.

Ploidy in bone marrow cells from healthy donors: a MAC (morphology antibody chromosomes) study.

The ploidy of human bone marrow cells belonging to the megakaryocytic, granulocytic-monocytic and erythrocytic lineages was studied by in situ hybridization using the biotin-labelled Y chromosome-specific DNA probe pY431 and the chromosome 1-specific probe pUC1.77 on cells identified morphologically and immunologically by the MAC procedure. Cells of the granulocytic-monocytic and erythrocytic lineages were seen to be 2N in ploidy, whereas the ploidy of the megakaryocytic lineage ranged from 2N to 32N, with the ploidy classes 4N and 8N being predominant. The frequency of megakaryocytes with 2N chromosomes was also high.

Amplification of fgfr4 gene in human breast and gynecological cancers.

We have investigated gene amplification of fibroblast growth factor receptor-4 (FGFR4) gene in 30 primary breast tumor samples and 15 gynecological tumor samples. Ten percent of the breast tumors showed 2- to 4-fold amplification. Amplification was found more frequently in estrogen- and progesterone-receptor-positive tumors and in tumors with high lymph-node involvement. Breast tumor samples were also analyzed for the amplification of fgfr3 and erbB2 genes and the chromosome 11q13 located genes hst1/int2/bcl1/sea. erbB2 gene was amplified 2- to 13-fold in 13% of the cases, but no amplification of int2/hst1/bcl1/sea amplicon was found. Gynecological tumors were also analyzed for the amplification of fgfr4 and fgfr3 genes and for int2 and hst1 oncogenes. Eleven of the 15 gynecological tumors were ovarian neoplasms including 2 benign tumors; the remainder comprised 1 ovarian metastasis of breast cancer; 1 endometrial cancer; 1 uterine leiomyosarcoma and 1 carcinosarcoma of the fallopian tube. In gynecological tumors, fgfr4 gene was found to be amplified in 2 ovarian tumors. Amplification of hst1 was found in 1 benign ovarian tumor. Thus, the fgfr4 gene may be involved in breast and ovarian tumorigenesis.

Cell lineage involvement of recurrent chromosomal abnormalities in hematologic neoplasms.

Analysis of most hematologic neoplasms indicates the involvement of one or more cell lineages in the bone marrow and/or the blood but rules out the involvement of all lineages in any one neoplasm. It is important to detect lineage involvement in order to clarify which stem cells are involved in leukemia, to predict prognosis, and to select appropriate treatment. Our aim was to study the cell lineage involvement of some of the recurrent chromosomal abnormalities seen in hematological neoplasms. The direct morphology-antibody-chromosomes (MAC) method was used. The deletion 20q in myeloproliferative diseases (MPD), the deletion of 5q and t(1;7) in myelodysplastic syndromes (MDS), and t(3;3) in acute myeloid leukemia subtype M7 (AML-M7) were seen in all or at least in two myeloid lineages. These were interpreted as stem cell abnormalities. Deletion 13q in MPD, t(8;21) in AML-M2 and t(15;17) in AML-M3 were seen in granulocytic lineages only; t(14;18) in non-Hodgkin's lymphoma and trisomy 12 as the sole abnormality in chronic lymphocytic leukemia (B-CLL) were seen only in immunoglobulin light chain clonal B cells; inversion 14 in T-CLL was seen only in T cells, whereas t(15;14) in acute lymphocytic leukemia with eosinophilia (ALL-EO) was seen in lymphoid stem cells but not in mature granulocytes or lymphocytes. Additional abnormalities (in addition to the Philadelphia chromosome) in chronic myeloid leukemia (CML) were seen in all myeloid cell lineages and also in mature granulocytes, B cells, and large granular lymphocytes. Abnormalities in Hodgkin's disease were restricted to CD30-positive Reed-Sternberg cells. Trisomy 8 and monosomy 7 are abnormalities that may be present in either stem cells or any of the single cell lineages.