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Michael E Nielsen

Publications and source records attributed to Michael E Nielsen.

7 recordsLinked to original sources
PubMed2006

Complement expression in common carp (Cyprinus carpio L.) during infection with Ichthyophthirius multifiliis.

A real-time PCR assay for determination of the complement response to infection with the ectoparasite Ichthyophthirius multifiliis in carp is presented. Specific primers were designed for selected genes representing the three pathways of the carp complement system. The investigated complement molecules were C1r/s, C3, C4, C5, factor I, factor B/C2-A (Bf/C2-A), mannose-binding lectin (MBL) and MBL-associated serine protease (MASP). The expression of the selected genes was analyzed on RNA extracts from skin, liver, and whole blood from carp at 3, 12, 24, 36, and 48 h post-infection (pi) with I. multifiliis. A pronounced up-regulation of Bf/C2-A, in skin, blood, and liver (250-, 60-, and 4-fold respectively), was observed at later sampling points pi (24-48 h). In addition, an intermediate (from 5 to 13-fold) down-regulation of MASP was observed in skin and liver samples at 36 and 48 h pi with respect to control fish. MBL was expressed only in liver and no variation in the transcription level of this lectin was observed. Complement factor C3 was significantly up-regulated in liver (4-fold up-regulation, 24 h pi). The presented results indicate that infection with the parasite I. multifiliis in carp to a large extent stimulates the expression of complement molecules. Moreover, the dramatic and early up-regulation of Bf/C2-A in skin indicates a role of this molecule as an acute-phase reactant. Furthermore, our study confirms the role of fish skin as an important extra-hepatic site of expression of complement molecules as well as an active regulator of complement expression. Expression of some of the components of the complement system in blood suggests that leukocytes in carp act as an important extra-hepatic source of complement molecules.

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PubMed2006

Ichthyophthirius multifiliis infection induces massive up-regulation of serum amyloid A in carp (Cyprinus carpio).

A real time quantitative PCR (RQ-PCR) assay was developed for measurement of differential expression of the genes encoding the acute phase reactant serum amyloid A (SAA), transferrin (TF) and a C-type lectin molecule (CL) in skin, blood and liver from Cyprinus carpio following infection with the ectoparasite Ichthyophthirius multifiliis. Serum amyloid A and CL were constitutively expressed in all organs evaluated while TF transcripts were only detected in the liver. A dramatic up-regulation (1600 times) in the expression levels of SAA was observed in skin 36 h after the parasite infection. A similar increase in the number of RNA molecules encoding for SAA was observed in the liver. The CL expression was significantly down regulated in all the organs and no significant change was observed in the expression levels of the TF in the liver. These results indicate that SAA plays a major role in the acute phase response in fish infected with I. multifiliis and emphasize the importance of the fish skin as an active organ in response to an ectoparasite infection.

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PubMed2006

Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses caused by the ectoparasite Ichthyophthirius multifiliis.

Real time quantitative PCR (RQ-PCR) assays were developed for the measurement of differential real-time expression of immune-related genes in skin and whole blood from Cyprinus carpio during an infection with the ectoparasite Ichthyophthirius multifiliis. The target genes included the chemokines CXCa and CXCb, the chemokine receptors CXCR1 and CXCR2, the pro-inflammatory cytokines interleukin 1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha) and the enzymes inducible nitric oxide synthase (iNOS) and arginase 2. The strongest up-regulation in skin was observed in the IL-1beta, CXCR1 and iNOS genes at 36-48h post-exposure to theronts. A significant up-regulation of the genes CXCa and TNF-alpha was also observed. An up-regulation of the expression of the genes CXCa, CXCR1, IL-1beta and iNOS was likewise found in blood, although the increase in the expression levels was more moderate and the expression peak was detected earlier in comparison with the skin. In addition, CXCR2 and the arginase 2 genes were specifically induced in blood. Our results confirm the role of CXCR1 and IL-1beta as two prominent molecules involved in the initiation of the inflammatory process in fish in relation to an ectoparasite infection. Moreover, this study confirms the role of carp skin as an important source of pro-inflammatory molecules as well as an active modulator of the local inflammation. Finally, expression and regulation of the evaluated genes in blood confirm the important role of the migrated leucocytes in the immune response against I. multifiliis.

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PubMed2006

Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses to injury mimicking infection with ectoparasites.

We studied a predictive model of gene expression induced by mechanical injury of fish skin, to resolve the confounding effects on the immune system induced by injury and skin parasite-specific molecules. We applied real time quantitative PCR (RQ-PCR) to measure the expression of the pro-inflammatory cytokines CXCa, CXCb, interleukin (IL)1-beta, tumor necrosis factor alpha (TNFalpha), and the receptors IL1R1, CXCR1 and CXCR2 in skin of Cyprinus carpio after mechanical injury. We also studied the expression of the anti-inflammatory cytokine IL-10. Most obvious, specific up-regulation of the chemokine CXCa, the chemokine receptor CXCR1 and the pro-inflammatory cytokine IL-beta was detected at 2-3h after injury. In order to correlate gene expression patterns after injury with cell migration, we studied chemotaxis of head kidney leukocytes towards lysates of epithelioma papulosum cyprini (EPC) cells. Neutrophilic granulocytes were shown to migrate towards epithelial lysates. Using immunohistochemistry we observed that the early inflammatory response after injury involved an influx of cells, most probably neutrophilic granulocytes, into the injured area. This suggests that the increased expression of pro-inflammatory genes is related to a rapid influx of neutrophilic granulocytes.

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PubMed2004

Simultaneous detection of marine fish pathogens by using multiplex PCR and a DNA microarray.

We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with < 20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.

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PubMed2002

Zebrafish as an immunological model system.

Two decades of research have established the zebrafish (Danio rerio) as a significant model system for studying vertebrate development and gene structure-function relationships. Recent advances in mutation screening, the creation of genomic resources, including the Zebrafish Genome Project and the development of efficient transgenesis procedures, make this model increasingly attractive for immunological study.

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