Biomedical subjects

Ysabel Santos

Publications and source records attributed to Ysabel Santos.

4 recordsLinked to original sources

PubMed2006

Cutaneous immune responses in the common carp detected using transcript analysis.

In order to detect new immune-related genes in common carp (Cyprinus carpio L.) challenged by an ectoparasitic infection, two cDNA libraries were constructed from carp skin sampled at 3 and 72h after infection with Ichthyophthirius multifiliis. In a total of 3500 expressed sequence tags (ESTs) we identified 82 orthologues of genes of immune relevance previously described in other organisms. Of these, 61 have never been described before in C. carpio, thus shedding light on some key components of the defence mechanisms of this species. Among the newly described genes, full-length molecules of prostaglandin D2 synthase (PGDS), the CC chemokine molecule SCYA103, and a second gene for the carp beta(2)-microglobulin (beta(2)m), beta(2)m-2, were described. Transcript amounts of the genes PGDS, interferon (IFN), SCYA103, complement factor 7 (C7), complement factor P (FP), complement factor D (FD) and beta(2)m-2 were evaluated by real-time quantitative PCR (RQ-PCR). Samples from skin, blood and liver from fish challenged with I. multifiliis were taken at 3, 12, 24, 36 and 48h post infection. Higher expression levels of most of these transcripts were observed in skin from uninfected fish, compared to the transcript levels detected in blood and liver from the same animals. Also, there was significant down-regulation of the genes PGDS and beta(2)m-2 in skin, whilst significant up-regulation was observed for the C7 and SCYA103 genes in liver of fish infected with the parasite. These results confirm the active role of fish skin in the immune response against infections, acting as an important site of expression of immune-related molecules.

Amino Acid Sequence↗

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.

Animals↗

PubMed2003

Development of a PCR-based method for the detection of Listonella anguillarum in fish tissues and blood samples.

A PCR assay for detection and identification of the fish pathogen Listonella anguillarum was developed. Primers amplifying a 519 bp internal fragment of the L. anguillarum rpoN gene, which codes for the factor sigma54, were utilized. The detection limit of the PCR using L. anguillarum pure cultures was approximately 1 to 10 bacterial cells per reaction. For tissue or blood samples of infected turbot Scophthalmus maximus, the detection limit was 10 to 100 L. anguillarum cells per reaction, which corresponds to 2 x 10(3) to 2 x 10(4) cells g(-1) fish tissue. Our results suggest that this PCR protocol is a sensitive and specific molecular method for the detection of the fish pathogen L. anguillarum.

Animals↗

PubMed2002

Reelin immunoreactivity in the larval sea lamprey brain.

In order to analyze the presence of a reelin-like protein in the brain of a primitive vertebrate with a laminar-type brain, such as the sea lamprey, Western blot and immunohistochemical approaches were employed by using the G10 and 142 reelin-specific monoclonal antibodies. Western blots of lamprey brain extracts showed bands of about 400 kDa, 180 kDa and others below 100 kDa; similar bands were observed in samples from rat cerebellum. In different larval stages there was a prominent reelin immunolabeling associated with the olfactory bulb, pallial regions, habenula, hypothalamus and optic tectum. In addition, the olfactory and optic tracts, as well as the afferent and efferent (fasciculus retroflexus) tracts of the habenular ganglion, also showed immunopositivity in these stages. Interestingly, the highest level of labeling was observed in premetamorphic larvae, just prior to entering the metamorphic stage. These data indicate that reelin expression is also prominent in brains of primitive vertebrates without layered cortical regions, suggesting that some physiological roles of reelin not related to the regulation of neuronal migration in layered cortical regions (i.e. involvement in axon pathfinding, synaptogenesis, dendritic arborization and neuronal plasticity) might have appeared earlier in evolution.

Animals↗