producing high-titer antibodies against a wide variety of antigens. CFA comprises of inactivated mycobacteria emulsified in
mineral oil. CFA is administered for the initial immunization,
while incomplete Freund’s adjuvant (IFA), which lacks mycobacteria, is used for subsequent immunizations [16].
3. Although CFA is commonly used in animal experiments, the
efficacy of leptospiral vaccines must also be determined using
adjuvants approved for human use. Other adjuvants, such as
aluminum hydroxide and aluminum phosphate, are less toxic
and therefore more acceptable in human preparations [17].
4. Female hamsters are conventionally used in Leptospira animal
studies because they are less aggressive than males. However, a
recent study suggests that male hamsters are more susceptible
to leptospirosis [18] compared to females infected with the
same number of bacteria. With this result, along with the US
National Institutes of Health’s policy that recommends the use
of animals of both sexes [19], performing vaccine studies with
both male and female hamsters in one group should be
considered.
5. CFA is a potent inflammatory agent. Accidental splashing in
the eyes with CFA can cause profound sloughing of skin or loss
of sight. Inadvertent needle punctures in humans can cause a
positive TB test and can result in inflammatory lesions and
abscess formation in tuberculin-positive individuals
[20]. Wear safety glasses and gloves when handling CFA.
6. To prevent denaturation of conformational epitopes, antigenCFA (or antigen-IFA) emulsions are prepared immediately
before immunization. Because heat is generated during the
emulsion preparation process, syringes should be laid on ice
to keep the emulsion close to 4
C. Some volume will be lost in
the mixing process; the volumes described here produce sufficient amount to immunize up to 15 hamsters.
7. Perform retro-orbital bleed under general anesthesia. One
hundred to 200 μl of blood is collected from the venous plexus
using a capillary tube. The eye is made to bulge, and the
capillary is inserted into the plexus with a gentle pressure and
a small rotation of the capillary. The blood is allowed to flow
into the capillary and is then transferred into a dry tube. After
blood collection, place a dry cotton pad over the eye with
gentle pressure to prevent retro-orbital hemorrhage and then
apply an ophthalmic ointment. Perform only one bleed per
week and alternate eyes to allow the tissue to heal. Monitor
animals for signs of ocular damage including bulging, squinting, swelling, and eye discharge following the procedure. If an
eye is damaged from the procedure, that eye may not be used
again for blood collection.
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Karen V. Evangelista and Kristel Lourdault
mineral oil. CFA is administered for the initial immunization,
while incomplete Freund’s adjuvant (IFA), which lacks mycobacteria, is used for subsequent immunizations [16].
3. Although CFA is commonly used in animal experiments, the
efficacy of leptospiral vaccines must also be determined using
adjuvants approved for human use. Other adjuvants, such as
aluminum hydroxide and aluminum phosphate, are less toxic
and therefore more acceptable in human preparations [17].
4. Female hamsters are conventionally used in Leptospira animal
studies because they are less aggressive than males. However, a
recent study suggests that male hamsters are more susceptible
to leptospirosis [18] compared to females infected with the
same number of bacteria. With this result, along with the US
National Institutes of Health’s policy that recommends the use
of animals of both sexes [19], performing vaccine studies with
both male and female hamsters in one group should be
considered.
5. CFA is a potent inflammatory agent. Accidental splashing in
the eyes with CFA can cause profound sloughing of skin or loss
of sight. Inadvertent needle punctures in humans can cause a
positive TB test and can result in inflammatory lesions and
abscess formation in tuberculin-positive individuals
[20]. Wear safety glasses and gloves when handling CFA.
6. To prevent denaturation of conformational epitopes, antigenCFA (or antigen-IFA) emulsions are prepared immediately
before immunization. Because heat is generated during the
emulsion preparation process, syringes should be laid on ice
to keep the emulsion close to 4
C. Some volume will be lost in
the mixing process; the volumes described here produce sufficient amount to immunize up to 15 hamsters.
7. Perform retro-orbital bleed under general anesthesia. One
hundred to 200 μl of blood is collected from the venous plexus
using a capillary tube. The eye is made to bulge, and the
capillary is inserted into the plexus with a gentle pressure and
a small rotation of the capillary. The blood is allowed to flow
into the capillary and is then transferred into a dry tube. After
blood collection, place a dry cotton pad over the eye with
gentle pressure to prevent retro-orbital hemorrhage and then
apply an ophthalmic ointment. Perform only one bleed per
week and alternate eyes to allow the tissue to heal. Monitor
animals for signs of ocular damage including bulging, squinting, swelling, and eye discharge following the procedure. If an
eye is damaged from the procedure, that eye may not be used
again for blood collection.
266
Karen V. Evangelista and Kristel Lourdault