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For the three experiments described later, the protocol for the first inoculation was identical.
In their study of changing Leishmania-susceptible BALB/c mice to Leishmania-resistant mice, they showed that the amount of parasites used for the first inoculation must be less than 3.3 × 10 promastigotes per mouse for the mice to build up a Th1 response.
Three adult Balb/c mice (12 week-old) were immunized three times intraperitoneally at 3-week intervals with 15 μg of purified HEV capsid protein, in the presence of complete Freund's adjuvant for the first inoculation, and incomplete adjuvant for the following immunizations.
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We then sequenced the ITS-5.8S rDNA sequences of the re-isolated fungus from the roots inoculated with S1 as well as the mtLSU sequences of the re-isolated fungus from the S3-inoculated roots and verified that these fungi were the same as those used for the first inoculations (data unpublished).
Blood samples (used for DNA and serum isolation) were collected weekly for 10 weeks after the first inoculation.
Seven to ten days after the first inoculation, all the plants were inoculated for a second time as above.
After challenge, Calf V shed the bacterium for a similar period as after the first inoculation; approximately 11 days.
For CVYV, plants were inoculated a second time five days after the first inoculation.
While dogs were used as sources of the virus for vaccine production, ferrets continued to be used as the source of the live virus for the second inoculation.
The animals were then sacrificed, 45 days after the first inoculation, to isolate splenocytes for lymphocyte proliferation and cytokine profiles in response to prime boosted antigen.
Experts recommend the first inoculation when a child is around 13 months old.
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