This method can help to answer key questions in the field of tuberculosis such as what is the role of adaptive immune responses and the pathogenesis of this multifactorial disease. The main advantage of this nucleic acid base method is that both bacterial loads and gene expression levels can be measured from the same individual. To begin this procedure, culture M.marinum on a 7H10 plate as outlined in the text protocol.
Transfer 10 milliliters of 7H9 medium containing ADC enrichment, polysorbate 80, and glycerol to a cell culture flask. Then, use a sterile one microliter inoculation loop to aseptically transfer one loopful of the M.marinum bacterial mass into the flask. Leave the cap loose or use a filter cap to allow for sufficient gas exchange and culture at 29 degrees Celsius in the dark without shaking for three to four days until the OD600 reaches about 0.7.
Dilute and continue culturing the bacteria as outlined in the text. To begin preparing the bacterial solution for infection, transfer one milliliter of the culture into a fresh tube. Centrifuge at 10, 000 g for three minutes.
Remove the supernatant and resuspend the pellet in one milliliter of sterile PBS. Using sterile PBS with a 0.3 milligrams per milliliter of phenol red as a tracer, dilute the suspension to reach the desired bacterial concentration. After this, use a one milliliter syringe to slowly pull the suspension through a 27 gauge needle three times.
Perform this step just before use for each aliquot. First, pipette a five microliter droplet of the diluted bacterial solution onto a piece of paraffin film. Then, pull the droplet into a 30 gauge insulin needle.
Use a five to eight-month-old fish for this experiment with one being a wild type fish and the other being a rag mutant fish. Position these fish ventral side up in the slits of a piece of moist foamed plastic. Inject the insulin needle between the pelvic fins at a 45 degree angle.
Keep the needle opening upwards to ensure that the entire opening is inside the abdominal cavity. Then, slowly inject the bacterial solution. After this, carefully remove the needle and immediately transfer the fish to a recovery tank filled with fresh tank water.
Take samples from the bacterial aliquot in use every 15 minutes on 7H10 plates. Incubate these samples at 29 degrees Celsius for five days to verify the infection dose. Check the wellbeing of the fish regularly, making sure to euthanize any fish with infection symptoms by incubating them in water with more than 0.02%of 3-aminobenzoic acid ethyl ester.
After euthanizing the fish, insert a pin posterior to the branchiostegal rays. Insert a second pin through the tail to tack the fish onto the platform. Using a scalpel, open the whole abdominal cavity.
Then, use a small spoon and sharp-ended tweezers to collect the internal organs starting at the heart and working along the spine towards the tail to detach all of the internal organs in one block. Use tweezers to detach the gut net from the cloaca and transfer the organs into a 1.5 milliliter homogenization tube containing half a dozen 2.8 millimeter ceramic beads. Immediately place the tube on dry ice to freeze the sample.
Store the sample at negative 80 degrees Celsius until ready to homogenize. After extracting the DNA and RNA, measure the mycobacterial loads by quantitative PCR as outlined in the text protocol. In this study, adult zebrafish are infected with M.marinum by an intraperitoneal injection.
A high infection dose is seen to lead to a progressive disease in which the mycobacterial load continues to increase until the average load reaches about five million bacteria ultimately killing the fish. A low dose on the other hand leads to the development of a disease spectrum similar to that seen in human tuberculosis with progressive, latent, reactivated, and sterilized infections. In this case, the load continues to increase for four weeks after which the disease reaches a steady state in the majority of the fish.
This data reveals that the initial number of mycobacteria used to infect the fish is a critical determinant for the outcome of the infection. Rag mutant zebrafish are seen to be unable to sufficiently limit the growth of mycobacteria leading to higher bacterial loads and increased morbidity. This clearly demonstrates the importance of adaptive immunity in controlling mycobacterial infection.
The levels of interleukin-4 are significantly higher in the wild type group revealing that the adaptive response is required for the efficient introduction of interleukin-4, but dispensable for the introduction of interferon gamma after mycobacterial infection. This protocol enables the collection of both DNA and RNA from the same sample which makes it possible to combine the mycobacterial burden of the sample with gene expression data of both the host and the bacteria. This technique can be combined with the administration of drugs or vaccine candidates to assess their effect on the mycobacterial loads and immune responses.
While attempting this procedure, it is important to remember to follow the local guidelines for personal safety and waste disposal when working with a biosafety level two pathogen. This technique makes it possible to study both active and latent tuberculosis with dormant mycobacteria in the zebrafish which is an ethical non-mammalian in vivo model.
