Hello! I want to know if there is a better way to detect the presence of a live attenuated vaccine in animal blood samples. We use the buffy coat fraction to extract the DNA and perform a PCR analysis to detect the genome of the vaccine (B. abortus RB51), but I still wonder if there is another (or better) way to do it (for example, extracting DNA from the whole blood sample).
Any recommendation?
Thank you!
You can simply culture the blood.
If the live attenuated vaccine strain such as Salmonella or E coli can be cultured on agar plates (containing appropriate media), then you can just isolate blood with anticoagulant and then spread about 100-200 ul blood directly on the plate.
If you had live organisms then you will observe respective bacterial colonies.
I don't think buffy coat DNA is a good idea, because the buffy coat contains white blood cells which have a certain density due to which they accumulate at the interface of the density media and plasma. However, bacteria may have higher density and thus may settle down with RBCs. SO you may not get DNA out of it.
If you still want to do DNA based PCR, you can isolate DNA directly from blood, without segregating the PBMCs. There are commercial kits available for isolation of DNA from blood.
God luck
S
Measure the increase in antibodies during spaced intervals and measurement CD4
It depends on whether you are trying to determine how much live attenuated B. abortus is in the blood or whether you are trying to determine the effect of vaccination (antibodies). I assume that you are trying to quantify the amount of live B. abortus in the blood. If that is the case, you can spread whole blood onto agar plates that select for the B. abortus and count the colonies. If you wish to detect all B. abortus (live and dead) you can use PCR on the serum. That works just fine. By taking the buffy coat you are missing some of the organisms.
you can detection of specific Antigen (to your vaccine) detection concentration by ELISA or detection total antibody (IgG & IgM) after & before vaccination
The main working princip of vaccines is to elicit specific immunological responses.If you know the type of vaccine you are looking for,its not dificult to design a test to detect and meassure the specific immnunological responses to the vaccine! Mainly you will be able to find out specific antibodies to the vaccine antigens and also certain type of celular responses for many of the possible vaccines! Do you know the vaccine you want to detect?
Gustavo
Best way to detect the presence of attenuated vaccine is ELISA. If you know which vaccine you are interested for, then you will easily get details of ELISA kit or antibodies. You can also refer guideline for testing of particular vaccine, there you will get details of testing of that vaccine.
Brucella abortus RB51 is a rough mutant strain used for vaccination in large animals. You can confirm presence of vaccine strain in blood by multiple ways
1. You can extract DNA from serum and subject to Bruce ladder PCR for confirmation. You can use commercial kits or laboratory methods for getting DNA from serum.
2. Isolation is gold standard for Brucella diagnosis and confirmation.
You can isolate Brucella by Blood culture methods. Castaneda biphasic culture method is mainly prescribed for isolation of B. melitensis from humans. It is a classical bacteriological method used for isolation of fastidious organisms. You need to prepare agar slant in bottles and add broth media to it for preparing biphasic medium. You can inoculate anti-coagulated blood sample and incubate it at 37 till bacterial growth is observed (it takes 7 days or long because Brucella is slow growing)
Another technique is lysis centifugation which involves culturing of buffy coat in serum supplemented media (Brucella can be isolated from lymphocytes because it is intrcellular pathogen)
You can try BACTEC blood culture system which is commercially available. (although it is mainly used for human blood smaples)
If you manage to get some colonies you can confirm it by culturing on blood agar. non hemolytic rough colonies could be B. abortus RB51
3. As many people said you can use ELISA to detect antibodies against RB51
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