I would like an efficient protocol so that the infectivity of leishmania donovani is maintained.
Dear Mohd Arish
Up to my knowledge the following protocol is good for maintenance of L.donovani.
Leishmania promastigotes were adopted and maintained in medium 199 pH 7•4 containing 0•025 M HEPES buffer and 10% (v/v) heat inactivated fetal bovine serum at 22°-26°C. Approximately 2 ×106 cells/mI promastigotes were inoculated in the medium and late log phase cells were harvested after 4 days of growth at 22°-26°C .
But you have to keep in your mind that continuous culture over time induces loss of virulence. If that happens, strains have to be maintained in golden hamster in the laboratory.
Reference:
http://www.ias.ac.in/jarch/jbiosci/19/291-299.pdf
Thanks for the suggestion but i want to know whether there is another way of culturing leishmania strains without maintaining them in golden hamster or balb/c mice.
Up to my knowledge there is a good way to preserve L.donovani promastigotes for 6-7 months by using blood agar slants. But more than this period of time I think that you have to use golden hamsters to activate the strain.
you can see the following reference
Muniaraj, M., Gupta, A. K., Narayan, S., Singh, D., Sinha, P. K., Kishore, K., ... & Bhattacharya, S. K. (2005). Preservation of Leishmania donovani promastigotes in blood agar slants. The journal of communicable diseases,37(3), 191-195.
Dear Dr. Mohd Arish,
We used the protocol described in the paper cited bellow with excellent results for culturing DD8 strain of Leishmania (Leishmania) donovani.
Species assignation of Leishmania from human and canine American tegumentary leishmaniasis cases by multilocus enzyme electrophoresis in North Argentina.
Marco JD, Barroso PA, Calvopiña M, Kumazawa H, Furuya M, Korenaga M, Cajal SP, Mora MC, Rea MM, Borda CE, Basombrío MA, Taranto NJ, Hashiguchi Y.
Am J Trop Med Hyg. 2005 May;72(5):606-11.
Best regards,
Diego Marco
In our laboratory we use liquid medium to grow up promatigotes. We have used M199 or RPMI or LIT or BAB. It is important to add human urine (2%) mainly to grow up L.infantum. To maintain the infectivity of the strain is important to inoculate in hamster.
In our lab we maintain the virulence by passage in hamsters (preferred) or mice. In order to avoid the animal maintenance all over the year, we isolate the parasite from spleen and freeze aliquots of the parasite after first (P1) or second (P2) passage. The virulence (either in vitro or in vivo) is quite excellent up to the fifth passage and still considerable after 10 passages. Whenever we want some parasite for challenge, we thaw the vial and perform the growth for one or two passages (so you may acquire the necessary amount of parasites).
hello
1-inoculate Amastigote from human blood in the abdomen of laboratory rats and after 2weeks take blood sample from rate and spleen and liver and bone marrow and detect the infection by R39 Stick or by ELIZA or PCR
2-For promastigote can be used NNN medium
my research about this work ...
for primary isolation use 3N, for propagation of the parasite use RPMI
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