8. Subcutaneous injection is administered on the scruff of the
hamster’s neck using a syringe with a 25 G needle. Use several
injection sites over the hamster’s back if larger volume of
vaccine is administered.
9. We only use low passage Leptospira challenge strain, i.e., passaged fewer than five times in vitro after isolation from a
mammalian host. Ensure the culture is in mid-log phase
(~1 Â 10
8 bacteria/ml) on the day of challenge.
10. Determine the density of Leptospira using a Petroff-Hausser
chamber. Dilute the Leptospira culture 1:100 in EMJH. Place
coverslip over the chamber in the center of the slide, and slowly
dispense 10 μl of diluted bacterial suspension under the coverslip. Allow the liquid to fill the chamber grid by capillary action.
Count bacterial cells within the center square (corresponding
to 25 small squares) under a dark-field microscope. Calculate
the density of Leptospira with the following equation: bacteria/
ml ¼ number of bacterial cells in all the 25 squares  dilution
factor  50,000. The value 50,000 is 50 (cell depth is
1/50 mm) Â 1000 (1000 mm
3
¼ 1 ml).
11. The ED 50 is the dose required for 50% of animals to meet the
endpoint criteria. Endpoint criteria include loss of appetite,
gait or breathing difficulty, prostration, ruffled fur, or weight
loss of >10% or maximum weight. ED 50 depends on the
serovar and the strain used and the number of in vitro passages.
L. interrogans serovar Copenhageni strain Fiocruz L1-130,
commonly used for hamster immunoprotection studies, has
an ED 50 of ~20 leptospires [21]. For the challenge, the use
of 1 Â 10
3 to 1 Â 10
4 (50–500 times the ED 50 ) has been
previously described [11, 21, 22].
12. Intraperitoneal injection is the most common inoculation
method for Leptospira studies [9]. However, IP inoculation
does not reflect the natural transmission of Leptospira. Other
routes of infection like through the skin (subcutaneous or
intradermal injection) [23] or through mucous membranes
(ocular inoculation) [24] that better simulate the natural
entry of the bacteria can be considered.
13. Serum must be prepared from the blood immediately after it is
collected by retro-orbital bleed. Because some antibodies lose
activity on repeated freezing and thawing, aliquot serum in
small volumes and store at À80
C.
14. Manual wash consists of filling the wells with PBS and emptying them three separate times. Emptying the wells by slapping
the plates against several layers of paper towels after each step
can minimize bubbles in the wells. Do not allow the wells to
dry. Avoid the formation of bubbles in wells because these may
Leptospirosis Vaccine Studies in Hamsters
267
hamster’s neck using a syringe with a 25 G needle. Use several
injection sites over the hamster’s back if larger volume of
vaccine is administered.
9. We only use low passage Leptospira challenge strain, i.e., passaged fewer than five times in vitro after isolation from a
mammalian host. Ensure the culture is in mid-log phase
(~1 Â 10
8 bacteria/ml) on the day of challenge.
10. Determine the density of Leptospira using a Petroff-Hausser
chamber. Dilute the Leptospira culture 1:100 in EMJH. Place
coverslip over the chamber in the center of the slide, and slowly
dispense 10 μl of diluted bacterial suspension under the coverslip. Allow the liquid to fill the chamber grid by capillary action.
Count bacterial cells within the center square (corresponding
to 25 small squares) under a dark-field microscope. Calculate
the density of Leptospira with the following equation: bacteria/
ml ¼ number of bacterial cells in all the 25 squares  dilution
factor  50,000. The value 50,000 is 50 (cell depth is
1/50 mm) Â 1000 (1000 mm
3
¼ 1 ml).
11. The ED 50 is the dose required for 50% of animals to meet the
endpoint criteria. Endpoint criteria include loss of appetite,
gait or breathing difficulty, prostration, ruffled fur, or weight
loss of >10% or maximum weight. ED 50 depends on the
serovar and the strain used and the number of in vitro passages.
L. interrogans serovar Copenhageni strain Fiocruz L1-130,
commonly used for hamster immunoprotection studies, has
an ED 50 of ~20 leptospires [21]. For the challenge, the use
of 1 Â 10
3 to 1 Â 10
4 (50–500 times the ED 50 ) has been
previously described [11, 21, 22].
12. Intraperitoneal injection is the most common inoculation
method for Leptospira studies [9]. However, IP inoculation
does not reflect the natural transmission of Leptospira. Other
routes of infection like through the skin (subcutaneous or
intradermal injection) [23] or through mucous membranes
(ocular inoculation) [24] that better simulate the natural
entry of the bacteria can be considered.
13. Serum must be prepared from the blood immediately after it is
collected by retro-orbital bleed. Because some antibodies lose
activity on repeated freezing and thawing, aliquot serum in
small volumes and store at À80
C.
14. Manual wash consists of filling the wells with PBS and emptying them three separate times. Emptying the wells by slapping
the plates against several layers of paper towels after each step
can minimize bubbles in the wells. Do not allow the wells to
dry. Avoid the formation of bubbles in wells because these may
Leptospirosis Vaccine Studies in Hamsters
267