Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo

Determination of bacteria concentration for inducing Immune response


  • Please log in to reply
14 replies to this topic

#1 Jevandrix

Jevandrix

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 01 June 2011 - 05:25 AM

Hi everyone,

I have more questions regarding my project that I would be grateful for your answers. I am currently have to work on inducing fish immune response through bacteria injection to select disease resistant fish for transcriptomic study. However one of the questions that bugs me is that what concentration of bacteria should I use. Is there any relevance of applying Lethal Dosage 50 for this?

If so, I have read journals on determining LD-50 by serial dilution and finding out which dosage kills 50% of the population, however the PhD student that I'm working with suggested to do ELISA at the end of the experiment . I do not quite understand what she meant. Since, she not very friendly, I don't want to annoy her. Thus, the help of the forum is much needed.

So, what is the standard practice in determining the concentration of bacteria to be used in inducing organism's immune response?

Thank you for your time and attention.

Much Obliged,
Jevandrix

#2 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 01 June 2011 - 06:16 AM

What you need is the following:

- a good book on the fish immunesystem (eg: the fish immune system:organism, pathogen and environmment, edited by G. Iwama and T. Nakanishi)
- a good book on fish pathogens in general (eg: bacterial fish pathogens: diseases of farmed and wild fish, 4the edition, by Austin B and Austin D).


I am not sure what that PhD student means with ELISA.
Maybe she means that she will test the death fish afterwars to see what the ELISA results are? You can use it to screen for antibody concentrations, but it seems weird to use it for a LD_50 dosage.

What you said, about the test is more accurate and used.

Anyway: there are bacteria out there that are able to kill fish even when there are only 10 bacteria present in that fish!
So you really need to know more about what bacteria you are working with, what they do etc...

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Jevandrix

Jevandrix

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 01 June 2011 - 06:47 AM

Dear Mr. Pito,

Thank you for your generosity, you can upload it at your convenience. This would certainly be a big help!

I would like to know your opinion on using LD-50 as the dosage to sufficiently induce immune response, is this the standard approach?

I am new to microbiology, I would certainly appreciate in guidance (books or links) to learn how to culture bacteria form stock to a desired concentration and understanding detailed correlation between absorbance, McFarlane and cfu.

Much Obliged,
Jevandrix

#4 PAO_ahac

PAO_ahac

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 94 posts
0
Neutral

Posted 01 June 2011 - 07:03 AM

suggested to do ELISA at the end of the experiment . I do not quite understand what she meant.

She suggested that you measure the production of any antibodies directed against the bacteria.

#5 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 01 June 2011 - 10:22 AM

suggested to do ELISA at the end of the experiment . I do not quite understand what she meant.

She suggested that you measure the production of any antibodies directed against the bacteria.

Of course, but how can you correlate this with the LD_50?

I would like to know your opinion on using LD-50 as the dosage to sufficiently induce immune response, is this the standard approach?

I am new to microbiology, I would certainly appreciate in guidance (books or links) to learn how to culture bacteria form stock to a desired concentration and understanding detailed correlation between absorbance, McFarlane and cfu.


LD_50 is just the dosage by which 50% dies, you can measure this by injecting different amounts (dilutions) in fish.
But I am not sure this is what you need since you need to have an immune response and transcriptomic study.
Do you need to know the LD_50 level? Or do you simply think that the dosages for LD_50 is big enough to get an immune response? Because if its the second one: lower dosages also induce an immune response..
But anyway for a lot of diseases this dosage is allready known, check the literature.
If you need standard microbiology knowledge, on how to dilute etc.. you should look for a general textbook like:
brooks microbiology or others.

I am not really sure what you want, are you simply doing a study on antibodies? Trying to find out which antibodies are formed against a certain bacterium? Or?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#6 Jevandrix

Jevandrix

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 01 June 2011 - 05:44 PM

Dear Mr. Pito,

I apologies if I have confused you. The idea here is to know what concentration of bacteria to be injected in fish to induce the immune response. Since a lot of studies done on determining LD-50 value on bacteria. I assumed that I can use this as a base for my choice of bacterial concentration.

I am not studying the antibody, but I'm studying what are the immune genes expressed by fish as a immune response towards the pathogen. However, since a lower dosage is significant to induce immune response, then, I might consider it.

I would definitely welcome a Fish Immunogenomics expert's additional opinion on this, are you one of them Mr.Pito?

Thank you Mr.Pito and Enthusiast for your help!


Much Obliged,
Jeevan

#7 leelee

leelee

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 652 posts
53
Excellent

Posted 01 June 2011 - 07:46 PM

I don't think using the LD50 dose would be at all appropriate for your work. In the first instance, using that dose will actually kill half of the fish you infect for your experiment- which is obviously very bad in terms of your results! In the second instance, I don't know much about working with fish, but surely you would not get ethics approval to use a dose that is pretty much guaranteed to kill 50% of your animals!

If I were you I would do the following-
1. literature search for articles published that used your particular fish, and the type of bacteria you want to use
2. figure out what dosage they used


Edit to add- before you do ANYTHING though. You need to take Pito's advice and go and do some reading of the basics.

Edited by leelee, 01 June 2011 - 07:48 PM.


#8 lab rat

lab rat

    Why does a science forum not have pictures of mice and rats?

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 245 posts
7
Neutral

Posted 01 June 2011 - 08:38 PM


suggested to do ELISA at the end of the experiment . I do not quite understand what she meant.

She suggested that you measure the production of any antibodies directed against the bacteria.

Of course, but how can you correlate this with the LD_50?


The fish that succumb to infection will not have sufficient time to produce adequate quantities of antibody, and will die of of acute infection. I am not versed in fish, but in mammalian immunology, the first, "general" Ab produced is IgM. The second, specific Ag is IgG (various classes). An individual with a recent infection will test positive for IgM via ELISA, while an individual with advanced or cleared infection will test positive for IgG. This is assuming that immunity is stimulated by a PAMP and not a toxin. Toxins may or may not stimulate an immune response (exo- vs endotoxin), and this type of immune response can diminish over time.

@the original poster: are you interested in the lethal dose, or the infectious dose? In the case of ID_50, you will see 50% of your population acquire the infection. You can measure the antibodies produced by symptomatic, asymptomatic infected, and uninfected individuals. This will be more likely to pass an IACUC audit, especially if you can reference the specific action of the pathogen and identify a humane endpoint for the study.

I can tell you that different types of fish have different levels of sophistication in their immune systems. Zebrafish are teleosts, and have been described as having an immune system similar to that of mammals.

Here are a few materials to read:
A.G. Zapata, A. Chiba and A. Vara. Cells and tissues of the immune system of fish. In: The Fish Immune System: Organism, Pathogen and Environment. Eds. G. Iwama and T.Nakanishi.
Zebrafish IFN receptor
Pubmed entry for Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: striking similarities and obvious differences with mammals.
Immunology and zebrafish: spawning new models of human disease.
42..."An immutable fixed-precision number of unlimited magnitude." <a href="http://en.wikipedia....amming_language)" target="_blank">http://en.wikipedia....amming_language)</a>, accessed 25June2009.

#9 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 02 June 2011 - 01:26 AM

I apologies if I have confused you. The idea here is to know what concentration of bacteria to be injected in fish to induce the immune response. Since a lot of studies done on determining LD-50 value on bacteria. I assumed that I can use this as a base for my choice of bacterial concentration.

I am not studying the antibody, but I'm studying what are the immune genes expressed by fish as a immune response towards the pathogen. However, since a lower dosage is significant to induce immune response, then, I might consider it.

AH ok, I see.

Its not about finding the LD_50 level.
I was confused with that.
Its just about getting the fish sick enough to produce antibodies.

If this is the case then you can use lower dosages , you need to check the literature for that.
Secondly: why not work with excisting vaccins? They stimulate the immunesystem of the fish just enough to make antibodies etc...

But you need to check that book I gave you: it explains the fish immunesystem in detail + not all fish are the same, as labrat said, its important to know.

Another thing: if you want to test for 1 specific disease you need to make sure the fish are ok, they need to be "sterile" in order to prevent they get another illness or something.
(altough, in your case this might not be a problem since you simple need the fish to respond to a bacterium.. it doesnt matter which one?)
I am not sure you are able to do this?
I have the feeling you (or that PhD student) do not have experience with working with fish and their pathogens because its not a simple set up to test.
You need to right set up, water quality and fish tank (because these are also parameters that can spike the fish immune system.. fish can become sick when using copper for example in your waterpipes), correct fish, paperwork from the governement, antibiotics etc...
Also: I do hope someone working with you is trained to work with fish? Had the propper training ? Because you cant just start up an experiment like this ... (normally you cant.. dont know where you live)

And about the ELISA: I think she simply means to use it to check for antibodies in general and when there are antibodies you can check the fish "immune genes" , btw: how are you going to look for these genes? Whats the set up? You do have certain information allready, because of this is not the case: I cant see you do this.
(check the second paper labrat gave you and you will immediately see that its not a simple set up.. you will need to screen lots and lots of geneproduces, rna etc)
You might want to check this:http://www.lifescied.org/cgi/reprint/5/3/287
Its not about fish, but its a general paper that might help you since its a set up for students to investigate genes correlating with immuneresponse




suggested to do ELISA at the end of the experiment . I do not quite understand what she meant.

She suggested that you measure the production of any antibodies directed against the bacteria.

Of course, but how can you correlate this with the LD_50?


The fish that succumb to infection will not have sufficient time to produce adequate quantities of antibody, and will die of of acute infection. I am not versed in fish, but in mammalian immunology, the first, "general" Ab produced is IgM. The second, specific Ag is IgG (various classes). An individual with a recent infection will test positive for IgM via ELISA, while an individual with advanced or cleared infection will test positive for IgG. This is assuming that immunity is stimulated by a PAMP and not a toxin. Toxins may or may not stimulate an immune response (exo- vs endotoxin), and this type of immune response can diminish over time.

@the original poster: are you interested in the lethal dose, or the infectious dose? In the case of ID_50, you will see 50% of your population acquire the infection. You can measure the antibodies produced by symptomatic, asymptomatic infected, and uninfected individuals. This will be more likely to pass an IACUC audit, especially if you can reference the specific action of the pathogen and identify a humane endpoint for the study.

I can tell you that different types of fish have different levels of sophistication in their immune systems. Zebrafish are teleosts, and have been described as having an immune system similar to that of mammals.

Here are a few materials to read:
A.G. Zapata, A. Chiba and A. Vara. Cells and tissues of the immune system of fish. In: The Fish Immune System: Organism, Pathogen and Environment. Eds. G. Iwama and T.Nakanishi.
Zebrafish IFN receptor
Pubmed entry for Acute phase response in zebrafish upon Aeromonas salmonicida and Staphylococcus aureus infection: striking similarities and obvious differences with mammals.
Immunology and zebrafish: spawning new models of human disease.


I would simply work with vaccins: there wont be any problems at all there for the AUDIT I think.
Since its not his intention to really investigate LD or ID.


Anyway: I still dont understand the ELISA linked with the LD_50, but I think this is misunderstanding from the start.

Edited by pito, 02 June 2011 - 01:33 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#10 Jevandrix

Jevandrix

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 02 June 2011 - 08:01 PM

Dear Mr. Pito & Mrs/Miss labrat,

I am overwhelmed with the supports I am getting. Thank you so much. I am currently reading the book that Mr. Pito gave. The fish is currently maintained by an aquaculture PhD student, it's currently stable and ready for challenge. The details of the research are as following:

Fish - Asian Seabass, Lates Calcarifer
Pathogen - Vibrio alginolyticus
Technology platform - RNA-seq and probably Digital Gene Expression (to study novel immune genes)
RTq-PCR (to study expression level of known immune genes)

As you can see, my part is to do the transcriptomic study for this set-up. The main target is to study disease-resistant fish (those who survived from the challenge) which is the natural resistance of the fish. So this leads me into questioning what concentration of V.alginolyticus to use. Should be sufficient enough to induce the immune response and lethal enough to be yielding strong disease-resistant fish?

I am currently reading literatures of similar studies carried on other fishes challenged with other pathogens. E.g, the recent paper on Lateolabrax Japanicos (Japanese Seabass) challenged with 0.2 ml of 1x 10^8 CFU injection of V. harveyi per fish. My first thought was -Where do they come up with this value?

From my tiny understanding, the ELISA would serve as a validation that the survived fish did survive through immune response.Am I right on this?

I am sorry for the redundancies, is just that, I am from Medical Molecular Genetics background with minute knowledge in fish-pathogen study.

Thank you for all of your guidance, I am looking at a lot of journals. Is just that I need to work fast and produce some results by end of July (at least the q-PCR part).

Much Obliged,
Jevandrix

#11 Jevandrix

Jevandrix

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 02 June 2011 - 08:10 PM

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

#12 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 03 June 2011 - 01:01 AM

check the following papers:

http://www.jeb.co.in...10/paper_25.pdf

http://docsdrive.com...009/509-513.pdf

http://en.cnki.com.c...NB200801024.htm

And this link: http://hotfile.com/d...2/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.


My first thought was -Where do they come up with this value?

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.


From my tiny understanding, the ELISA would serve as a validation that the survived fish did survive through immune response.Am I right on this?

yes, to see if there are antibodies.

The main target is to study disease-resistant fish (those who survived from the challenge) which is the natural resistance of the fish


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.



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


Sounds ok.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#13 Jevandrix

Jevandrix

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 03 June 2011 - 07:18 AM

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

#14 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,328 posts
80
Excellent

Posted 03 June 2011 - 08:40 AM

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


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.

Edited by pito, 03 June 2011 - 08:41 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#15 Jevandrix

Jevandrix

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 06 June 2011 - 08:26 PM

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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.