In past few months, I encountered a problem that the leishmania infection (L. amazonensis LV78 and L. donovani HU3) in peritoneal mouse macrophages (from BALB/c mice) in vitro became low. I used to infect macrophages with LV78 and HU3 promastigotes at 10:1 and 20:1, respectively for 24 hours and obtained 500 amastigotes per 100 macrophages after removing uninfected promastigotes followed by 72 hour incubation. Recently, without any changes, the number of amastigotes after 72 hours dropped a lot. I have checked that there are infection after incubating promastigotes with macrophages, implying that the amastigotes were rapidly cleared by the macrophages after infection. I have tried using peritoneal mouse macrophages from nude mice but the result was similar. Does anyone encountered similar phenomenon?
Have you think about isolating metacyclics (Ficoll gradient) ?
Are you sure you used the best moment of their growth (few days after the beginning of stationary phase) ? According to serum or passages number, you may have some differences.
If you need more parasites, you can centrifuge your macrophages and promastigotes (we do it 300xg for 5 min to help the parasites)
Are you sure about the infectivity of Leishmania strains? Maybe you will need to infect the animals to recover the highly infective parasites.
Have you changed the protocol to harvest peritoneal macrophages? Some drugs used to euthanize animals may interfere with the activation state of macrophages, especially when given intraperitoneally.
If you yielded few amastigotes that meat that most probably your problem is not related to the infectivity of the strain! May be you can try using immunoregulatory cytokines such as IL10. There are a few studies on that, search on google
Hi Chinfung Chan ,
1. The virulence of pathogen/parasite is the key mediator in the efficiency of infection.
2. If the parasites (promastiogotes) are less efficient in causing infection to macrophages then it could be due to less virulence.
3. Virulence can be recovered after in vivo passage.
4. One reason suggested by Débora Botura Scariot can be helpful in your case if you used any special drug in euthanizing the animals at the time of peritoneal macrophages isolation.
5. And you did not mentioned that at what temperature you are performing all these that can be another issue.
Thank you,
Good luck
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