Determination of bacteria concentration for inducing Immune response - The relevance of LD-50 in inducing immune response (Jun/01/2011 )

Dear Mr. Pito,

The immune genes that currently looking are both novel and known genes. After extraction of RNA from spleen, head kidney and thymus, I will send some for Next Generation Sequencing and some for RTq-PCR to detect the expression level of known genes. I have already checked the known immune related genes for Asian Seabass at NCBI database. Which are MHC, Apolipoprotein, Cethapsin, goose-type lysozyme, leukocyte cell-derived chemotaxin and Mx protein.

I will design the primer and evaluate them before running RT-PCR. This is the approach that I am focusing now to produce result fast and publication.

What's your opinion?

Thank You,
Jevandrix

check the following papers:

http://www.jeb.co.in/journal_issues/201007_jul10/paper_25.pdf

http://docsdrive.com/pdfs/medwelljournals/rjbsci/2009/509-513.pdf

http://en.cnki.com.cn/Article_en/CJFDTOTAL-HNNB200801024.htm

And this link: http://hotfile.com/dl/119727260/1d5aed2/aaqa.pdf.html
(its a book, check the chapter on gene expression, isotation and cloning, check the references there, it might help you too)

I think that those papers + the books will be a big help.

Ask the writers of that paper?
It could be experience, maybe from a former test? Maybe its the IC_50?

Anyway: I dont all the details about what concentration should be used. It also depends on the age, size of the fish etc.
You need to find a concentration that is big enough to start a reaction and vaccins are designed to do this, so if you can find or prepare one yourself, it is a good start.

Or just check the literature, the books I gave and look for concentrations of that specific pathogen that cause illness.. The main important thing is to make sure you dont use too much, you do not want to kill your fish.

yes, to see if there are antibodies.

I do not know what you mean by this?
Natural resistance, you mean that you wont be using antibiotics to cure the fish? or ...?


BTW: do keep in mind that those fish are kept in specific circumstances, maybe they are allready being given antibiotics to make sure they dont get sick....
This could destroy the entire research

Also: think about how you give them the bacteria... stress is a big factor.
But I suppose that the PhD student handeling the fish knows all of this and will guide you on that.

Sounds ok.

Dear Mr. Pito,

Thank you for your recent guidance. Regarding the disease-resistant fish; the aquaculture PhD student was explaining to me that it is more logical to study the natural resistance of (non-vaccinated) fish, in order to screen disease-resistant fish for selective breeding. This screening will be done by bioinformatics tool that will be developed based on my transcriptomic study.

So, he further explained that there will be mortalities among the live bacteria injected fish. However, there will be surviving fish, which will be the subject of the research. Subsequently, ELISA conducted, to confirm antibody production and later the transcriptome profiling of immune-related genes. He explained that vaccinating fish revolves around studying the efficiency of the vaccine. Corresponding bioinformatic product developed through my research can be used for downstream application such as this (vaccine efficiency validations).

This is the current big picture that I have on the research. I am eager to know your opinion.

Much Obliged,
Jevandrix

Jevandrix on Fri Jun 3 15:18:43 2011 said:

What I mean is: you can use the vaccin to provoke an immuneresponse.
ANd this way you dont kill the fish.
Altough, maybe the immune response is not strong enough to detect all the genes you are looking for (but its a start).


Just to be clear: the use of the vaccin is not something I proposed to study the efficiency or how to make one.. I proposed it because its a safe and easy way to get an immune response and they are commercially avaible, thus its easier for you to start with then starting with figuring out what concentrations you need to use etc.. Esp because you seem to have problems with the microbiological concepts of dilution etc...
+ have you concidered which adjuvant you will use when working with the live bacteria etc...
(vaccins are a lot easier.. they are prepped and ready to use...)

I also have the feeling that that PhD student has a second agenda .... namely the breeding or selecting of fish based on their response to the bacteria ...
And I think thats why they dont want you to work with a vaccin.. because they want to start selecting fish...
And if this is the case, then yes, you better work with live bacterium and with for example LD_50, IC_50 etc...

+ also : if its about selecting the "strongest" fish, make sure the parameters between the fish (tanks) are equal.. its not always about the "strongest-best immune" system.. sometimes is about other things like stress, feed etc.. so you need to fix those parameters etc.

Another thing: you mention you will be injecting the fish with the bacterium? This if of course again a completely different set up then when you work with bacterium added to the water etc...

You have a lot of parameters to take in act.

Anyway: I find it weird you dont have a supervisor or someone else to guide you with the set up of these trials ... because its a lot more complicated then just "making the fish sick" with a bacterium and see which fish survived and then screen them.

Dear Mr. Pito and everyone,

I really appreciate your guidance so far. I will try to study all the journals and books to gain an in depth understanding of what I have to do. I will keep posting about doubts that I'll have in the future.

Thank you so much for your time and attention. God bless you all.

Much Obliged,
Jevandrix