Validation of mucosal sampling and analysis - a 3R alternative to serum from killed fish
We have developed a method for gentle sampling of skin mucus from live fish. The method uses soft tissue paper placed on top of mucus to absorb and sample the liquid part of the mucus, leaving the "gel" intact on the skin. In the present experiment we will seek to validate and document the versatility of the method. The overall aim is to document how well this method compare with both serum samples and mucus samples obtained by less gentle methods.
From the 30 previously vaccinted fish going in to the experiment, we will take mucus samples using three different methods: gentle scraping of mucus using plastic scraper, wiping off of mucus using paper and finally absorption of liqud from mucus using tissue paper. Samples will be taken from individually tagged fish at different time intervals to estimate the recovery rate of mucus. From other fish mucus will be sampled from different parts of the fish (using absorption) to compare possible differences in mucus quality. Serum taken from fish at termination will be compared to various mucus samples and mucus sampling sites.
The mucus samples will be analysed by ELISA for specific antibodies against antigens in the vaccine they have previously received as well as for levels of complement C5. Mucus will also be analysed by proteomic and metabolomic methods. Using such diverse methods will allow us to evaluate how representative the mucus liquid is as for the fish.
From the 30 previously vaccinted fish going in to the experiment, we will take mucus samples using three different methods: gentle scraping of mucus using plastic scraper, wiping off of mucus using paper and finally absorption of liqud from mucus using tissue paper. Samples will be taken from individually tagged fish at different time intervals to estimate the recovery rate of mucus. From other fish mucus will be sampled from different parts of the fish (using absorption) to compare possible differences in mucus quality. Serum taken from fish at termination will be compared to various mucus samples and mucus sampling sites.
The mucus samples will be analysed by ELISA for specific antibodies against antigens in the vaccine they have previously received as well as for levels of complement C5. Mucus will also be analysed by proteomic and metabolomic methods. Using such diverse methods will allow us to evaluate how representative the mucus liquid is as for the fish.