Fish respond differently to different kind of challenge...its up to you to standardize and optimize the route of challenge and dose of challenge with a set of pathogenecity study with the pathogen and host you are using. With my experience, sea bass being a warm water species it should be easy to optimize an immersion challenge with a bacterial pathogen if not, i.p. or i.m are better methods.

It is true that IP/ IM challenge does not mimic the natural mode by which fish encounter pathogens. However, these methods may be tried to get the results in a short span of time. Otherwise, why not to try both IP and immersion / dip method and get a comparative result also.

Dear colleague, It is difficult to predict which approach is the best. You may have to measure the stress response itself (e.g. cortisol levels in blood) to determine which method gives minimal handling stress. It could help to anaesthetize (MS222) the animals before any further handling.

For any procedure in fish like immunization, bleeding,administration of drugs like an immunostimulant etc. if it is likely to cause stress, it is advisable to anaesthetize the fish (as Prof Muiswinkel suggested) before doing the procedure. we anaesthetize some fish species before challeging them intraperitoneally with a virulent bacterial pathogen.

Sure, anesthesia is very important and that is a factor we do not compromise. It is also on good record that the anesthesia procedure itself is stressful to fish, especially MS222, so we anesthetize them in cold water, at between 5-10*C and it works like magic (takes less than 2 mins for them to be knocked off). In the literature, you would notice that most marine species are challenged with pathogens through dipping, as such I would not be the first. I however wanted to get information from those experienced with this practice with regards to the time period from the challenge that is considered suitable to notice any cytokines in the blood, liver and kidney. I have read papers where it was reported that mortality is usually recorded from day 4 onwards when dip-challenged, whereas with IP challenge, fish mortality have been reported at within 24 hours onwards. So, if it would take a fish (challenged with 10^6 CFU of pathogens) 4 days to die, is it advisable to begin blood sampling for cytokines at 24 hrs or after 48 hrs or more, assuming cortisol level due to stress of anesthesia is kept constant?

This is a good question, I think that dipping is more better than IP. The later method equals infection with fungus, that is the result of injection.

Another point, I think that anasthesia is not good idea. You will measure some parameters in blood,. Do you think that the anesthetic compound will not affect or change you results?. Well, try with or without and compare.

Hello Ismail, do you mean to simply grab a live, dip-infected fish and try to collect blood? This would raise other issues;

1. It does not sound very ethical, our ethics committee could ban me if I were to do that. It would be considered cruelty

2. The struggling fish would make blood collection very challenging

3. The struggle of the fish would raise cortisol level and likely stimulate the release of the very cytokines I am hoping to measure due to infection

Anesthesia is to stabilize the fish to collect blood with minimal stress to the fish, or else the whole exp is compromised. So it is necessary.

Thank you for your comment. Basically, I am not against fish rights or ethics . Again, I do not advise to use chemical anasthetics especially MS222 in blood analysis.

I would like to add that even clapping with hands raise cortisol levels in fish blood.

There are different methods that not alter the blood consistituents other than .chemical anasthetics. Remember, that I asked you try with or without and compare.

Thank you again, hoping sucessful study.

Now we are on the same page, Ismail, no insults intended.

It is for the knowledge of interference of MS222 with blood chemistry that we opted for the use of temperature manipulations. With our fish, at 10-12*C, they simply go to sleep within 2 minutes and make full recovery within 15 min.We are in the process of checking the cortisol level based on the stress effect of this procedure and hopefully compare to that with MS222.

Further, I agree with you totally on the dipping procedure, as was corroborated by 2 other professors from Canada and Stirling.

Thanks again and best wishes to you as well.

Salam