Identification and genetic characterization of Saprolegnia parasitica, isolated from farmed and wild fish in Finland.

Oomycete infections in farmed fish are one of the most significant disease issues in salmonid aquaculture worldwide. In the present study, Saprolegnia spp. in different farmed fish species in Finland were identified, and the molecular epidemiology of especially Saprolegnia parasitica was examined. We analysed tissue samples from suspected oomycete-infected salmonids of different life stages from a number of fish farms, as well as three wild salmonids. From collected oomycete isolates, the ITS1, 5.8S and ITS2 genomic regions were amplified, analysed phylogenetically and compared with corresponding sequences deposited in GenBank. Of the sequenced isolates, 91% were identified as S. parasitica. Isolates of yolk sac fry were identified as different Saprolegnia spp. Among the isolates from rainbow trout eggs Saprolegnia diclina dominated. In order to determine potential dominating clones among the S. parasitica, isolates were analysed using Multi Locus Sequence Typing (MLST). The results showed that one main clone contained the majority of the isolates. The MLST analysis showed four main sequence types (ST1-ST4) and 13 unique STs. This suggests that the Saprolegnia infections in farmed fish in Finland are not caused by different strains originating in the farm environment. Instead, one main clone of S. parasitica is present in Finnish fish farms.

Detection and Quantification of the Oomycete Saprolegnia parasitica in Aquaculture Environments.

Saprolegnia parasitica induces heavy mortality in aquaculture. The detection of S. parasitica is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously published real-time quantitative PCR (qPCR) assay to confirm the presence of S. parasitica in fish and in water using environmental DNA (eDNA) quantification. Analytical sensitivity and specificity of the assay was assessed in silico, in vitro and the qPCR assay was compared with microbiological cultivation methods to detect and quantify S. parasitica in water samples from a controlled fish exposure experiment and from fish farms. Furthermore, we compared the use of an agar cultivation method and the qPCR assay to detect S. parasitica directly from mucus samples taken from the fish surface. The analytical sensitivity and specificity of the qPCR assay were high. The qPCR assay detected 100% of S. parasitica-positive water samples. In a field study, the qPCR assay and a microwell plate (MWP) enumeration method correlated significantly. Furthermore, the qPCR assay could be used to confirm the presence of S. parasitica in skin mucus. Thus, the qPCR assay could complement diagnostic methods in specifically detecting saprolegniosis in fish and used as a surveillance method for S. parasitica pathogen in aquaculture environments.

Potentiation of natural killer cell activity with myricetin.

BACKGROUND/AIM: Myricetin is a flavanoid that can be found in a variety of food sources, including red wine. Several reports have indicated that flavonoids may reduce the disease risk of cancer. The aim of the present study was to investigate the effect of some flavonols on natural killer (NK) cell activity. MATERIALS AND METHODS: A time-resolved fluorometric assay (TDA-labeled K562 target cells) was used for measuring the cytotoxic activity of NK-cells pre-treated with different flavonoids. RESULTS: A limited number of flavanoids was tested for their ability to enhance the NK activity. Pre-treating NK cells with myricetin, could potentiate their ability to kill K562 erythroleukemia cells. This enhancement of the NK activity was observed in a dose-dependent manner. Similar treatments with the structurally similar molecule quercetin, that lacks one hydroxyl group, did not have any impact at all on NK activity. CONCLUSION: The enhanced cytotoxic activity observed with myricetin-pretreated-NK cells might shed some light on human studies indicating a preventive role of flavonols against cancer.

Novel class of potent 4-arylalkyl substituted 3-isoxazolol GABA(A) antagonists: synthesis, pharmacology, and molecular modeling.

A number of analogues of the low-efficacy partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups, were synthesized and tested as GABA(A) receptor ligands. Substituents of different size and structural flexibility such as alkyl, phenylalkyl, diphenylalkyl, and naphthylalkyl were explored. Pharmacological characterization of the synthesized compounds was carried out using receptor binding assays and by electrophysiological experiments using whole-cell patch-clamp techniques. Whereas none of these compounds significantly affected GABA(B) receptor sites or GABA uptake, they did show affinity for the GABA(A) receptor site. While alkyl or benzyl substitution, compounds 7a-h, provided receptor affinities comparable with that of 5 (K(i) = 9.1 microM), diphenylalkyl and naphthylalkyl substitution, as in compounds 7m-t, resulted in a dramatic increase in affinity relative to 5. The 3,3-diphenylpropyl and the 2-naphthylmethyl analogues, compounds 7s and 7m, respectively, showed the highest affinities of the series (K(i) = 0.074 microM and K(i) = 0.049 microM). In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine (1), compounds 7m and 7s showing antagonist potency (IC(50) = 0.37 microM and IC(50) = 0.02 microM) comparable with or markedly higher than that of the standard GABA(A) antagonist 4 (IC(50) = 0.24 microM). Highly potent convulsant activity was demonstrated in mice with compounds 7m (ED(50) = 0.024 micromol/kg) and 7s (ED(50) = 0.21 micromol/kg) after intracerebroventricular administration, whereas no effects were found after subcutaneous administration. According to a previously proposed pharmacophore model for GABA(A) receptor agonists, a receptor cavity in the vicinity of the 4-position of the 3-isoxazolol ring in 4-PIOL exists. A molecular modeling study, based on compounds 7o,m,l,q,s, was performed to explore the dimensions and other properties of the receptor cavity. This study demonstrates the importance of the arylalkyl substituents in 7m and 7s and the considerable dimensions of this proposed receptor cavity.