Danio Rerio as Model Organism for Adenoviral Vector Evaluation

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectorsThis research was funded by Xunta de Galicia, Local Government, grant number ED431C 2018/28, and Gobierno de Navarra, Local Government, grant numbers 0011-1383-2018-000011 PT038 and 0011-1383-2019-000006 PT013 (XANTOGEN)S

A real-time quantitative PCR assay using hydrolysis probes for monitoring scuticociliate parasites in seawater

Scuticociliatosis is a serious disease that affects flatfish during culture and against which no effective control measures have yet been developed. Monitoring parasite levels in the water may be a valuable way of establishing the risk of infection and enabling appropriate control measures to be taken, thus representing an advance in controlling the disease. To achieve this objective, we have designed a quantitative real-time PCR (qPCR) assay using primers (f / r ITS2) and a hydrolysis probe that specifically amplify a region of the internal transcribed spacer 2 (ITS2) of the main aetiological agents of scuticociliatosis: Philasterides dicentrarchi and Miamiensis avidus. The slope (m), efficiency (E) and linearity (R2) determined from the standard curves generated are within the optimal values for qPCR. The high analytical sensitivity of the qPCR assay enables quantification of less than 120 pg of DNA per μL of reaction and detection of 1 ciliate per assay. The qPCR assay also exhibits high precision, with intra- and inter-assay coefficients of variation (CV) of respectively 0.27 and 7.57%. The protocol developed for isolating and quantifying ciliates seawater samples it has a recovery efficiency greater than 75% when the ciliate levels are between 103 and 2 × 103 ciliates/L and the turbidity of the water does not exceed one nephelometric turbidity unit (NTU). The real-time qPCR assay developed is a useful and appropriate tool for the specific and sensitive monitoring of scuticociliates in the water used in flatfish farms, enabling the establishment of effective prevention and control programmesThis study was financially supported by grants PID 2020-113087RB- I00 awarded by the Ministerio de Ciencia e Innovación (Spain) and the European Regional Development Fund (ERDF) (European Union), IDI-20200702 by Centro para el Desarrollo Tecnológico Industrial (CDTI) of the Ministerio de Ciencia e Innovación (Spain) and ED431C2021/26 from the Xunta de Galicia (Spain), and by the PARAFISHCONTROL project, which received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 634429. This publication only reflects the views of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained hereinS