2.7.6 Analysis
of the Immunization
Potential of p36p
À
Sporozoites
1. BALB/c mice.
2. C57BL6 mice.
3. p36p
À sporozoites or RAS or PBS.
4. Giemsa-stain.
5. Real-time PCR machine.
6. A-type 18S ribosomal RNA primers.
2.8 Gamma
Irradiation
for Mutagenesis
in Parasites [25]
2.8.1 Dissection
of Sporozoites
1. 50% Human plasma diluted in saline.
2. X-ray beam of a Picker Vanguard teletherapy unit.
3. A/J mice.
4. Injection needle.
5. Hamster.
6. Plasmodium berghei.
3 Methods
3.1 Bacterial
Virulence Attenuation
by Passaging [8]
M. bovis strain is subjected to culturing on potato slices for 13 years.
These potato slices are cooked in beef bile with glycerol supplementation. The strain obtained after passaging is found to lose
virulence. This strategy was employed in the development of the
BCG vaccine against tuberculosis.
3.2 Bacterial
Virulence Attenuation
by Mutagenesis
1. Isolation of galE mutants: S. typhi, strain Ty 2 is grown in brain
heart infusion (BHI) broth. Exponential growth phase culture
is plated onto Endo agar where lactose is replaced by galactose
by either directly or after mutagenic treatment with JCR-170,
N-methyl-N
0 -nitro-N-nitrosoguanidine (NG) or ultraviolet
light.
2. Stability Test: The stability of the mutants is tested by plating
on Endo agar either directly or after treatment with JCR-170
or NG. For in vivo stability test, Swiss white mice are inoculated
with 10
7 cells intraperitoneally. After 5–10 days, mice are
euthanized, and their liver and spleen are harvested. The harvested liver and spleen are homogenized in 0.85% NaCl in a
Potter-Elvehjem homogenizer. The homogenate is plated onto
galactose-Endo agar.
3. Galactose-induced bacteriolysis: Cells are grown in infusion
broth at 37
C on a rotating shaker at 142 rpm. Growth is
monitored by measurement of the optical density at 550 nm.
Galactose (0.1%) is added at the moment of inoculation or
when the culture is in the logarithmic phase of growth.
4. Enzyme assays: The procedures for the assays of galactosepermease and galactose-l-phosphate-uridyl transferase are as
Live Vaccine Attenuation
341
of the Immunization
Potential of p36p
À
Sporozoites
1. BALB/c mice.
2. C57BL6 mice.
3. p36p
À sporozoites or RAS or PBS.
4. Giemsa-stain.
5. Real-time PCR machine.
6. A-type 18S ribosomal RNA primers.
2.8 Gamma
Irradiation
for Mutagenesis
in Parasites [25]
2.8.1 Dissection
of Sporozoites
1. 50% Human plasma diluted in saline.
2. X-ray beam of a Picker Vanguard teletherapy unit.
3. A/J mice.
4. Injection needle.
5. Hamster.
6. Plasmodium berghei.
3 Methods
3.1 Bacterial
Virulence Attenuation
by Passaging [8]
M. bovis strain is subjected to culturing on potato slices for 13 years.
These potato slices are cooked in beef bile with glycerol supplementation. The strain obtained after passaging is found to lose
virulence. This strategy was employed in the development of the
BCG vaccine against tuberculosis.
3.2 Bacterial
Virulence Attenuation
by Mutagenesis
1. Isolation of galE mutants: S. typhi, strain Ty 2 is grown in brain
heart infusion (BHI) broth. Exponential growth phase culture
is plated onto Endo agar where lactose is replaced by galactose
by either directly or after mutagenic treatment with JCR-170,
N-methyl-N
0 -nitro-N-nitrosoguanidine (NG) or ultraviolet
light.
2. Stability Test: The stability of the mutants is tested by plating
on Endo agar either directly or after treatment with JCR-170
or NG. For in vivo stability test, Swiss white mice are inoculated
with 10
7 cells intraperitoneally. After 5–10 days, mice are
euthanized, and their liver and spleen are harvested. The harvested liver and spleen are homogenized in 0.85% NaCl in a
Potter-Elvehjem homogenizer. The homogenate is plated onto
galactose-Endo agar.
3. Galactose-induced bacteriolysis: Cells are grown in infusion
broth at 37
C on a rotating shaker at 142 rpm. Growth is
monitored by measurement of the optical density at 550 nm.
Galactose (0.1%) is added at the moment of inoculation or
when the culture is in the logarithmic phase of growth.
4. Enzyme assays: The procedures for the assays of galactosepermease and galactose-l-phosphate-uridyl transferase are as
Live Vaccine Attenuation
341