should also be included. For fitness testing in animals, the input
pool should include previously described mutants that are attenuated in their fitness such as loa22 [6, 13] and mutants with
wild-type fitness in tissues such as ligB [6, 14] or flaA1 [6, 15].
5. Although intraperitoneal inoculation is the most common
challenge route for studies of Leptospira infection [16], it
does not reflect the natural route of infection. Routes into or
through the skin (subcutaneous or intradermal inoculation),
respectively [17], or through mucous membranes (ocular inoculation) [11], which simulate the natural entry of the bacteria,
can be considered. The choice of challenge route depends on
the purpose and the design of the experiment. The volume of
inoculum needs to be adapted to the route of challenge. For
intraperitoneal challenge, the maximum volume that can be
injected is 1 ml, whereas only 10 μl can be used for ocular
inoculation [11]. Keep in mind that the more natural routes of
inoculation have not been examined for bottleneck effects,
which lead to stochastic rather than deterministic changes in
measured fitness levels.
6. The number of animals to use for each experiment depends on
the number of mutants in the input pool. This number can be
calculated by power analysis, which may require a pilot
experiment [7].
7. DNA must be extracted from the blood immediately after its
collection.
8. Selection of the organs to be harvested depends on the goals of
the experiment. Tissues that are the main sites of disease
pathology, such as kidney, liver, and lung, are often collected.
However it has been shown that Leptospira can disseminate to
and colonize many other organs [11].
9. The exact medium composition and incubation scheme
depend on the conditions being examined. The number of
replicates to be tested depends on the conditions being examined and the number of mutants in the pool. A pilot experiment with a small number of mutants may be necessary to
calculate the appropriate number of replicates from a power
analysis.
10. Carefully remove the supernatant; pellets are loose due to the
low-speed centrifugation.
11. Calculate the volume of tissue to use for DNA extraction with
the following formula: volume in μl ¼ (500 μl  25 mg)/
(mg of diced tissue). For example, 250 μl homogenate will be
used from a 50 mg tissue resuspended in 500 μl PBS.
12. Wear earmuffs to protect hearing.
Transposon Sequencing in Leptospira interrogans
105
pool should include previously described mutants that are attenuated in their fitness such as loa22 [6, 13] and mutants with
wild-type fitness in tissues such as ligB [6, 14] or flaA1 [6, 15].
5. Although intraperitoneal inoculation is the most common
challenge route for studies of Leptospira infection [16], it
does not reflect the natural route of infection. Routes into or
through the skin (subcutaneous or intradermal inoculation),
respectively [17], or through mucous membranes (ocular inoculation) [11], which simulate the natural entry of the bacteria,
can be considered. The choice of challenge route depends on
the purpose and the design of the experiment. The volume of
inoculum needs to be adapted to the route of challenge. For
intraperitoneal challenge, the maximum volume that can be
injected is 1 ml, whereas only 10 μl can be used for ocular
inoculation [11]. Keep in mind that the more natural routes of
inoculation have not been examined for bottleneck effects,
which lead to stochastic rather than deterministic changes in
measured fitness levels.
6. The number of animals to use for each experiment depends on
the number of mutants in the input pool. This number can be
calculated by power analysis, which may require a pilot
experiment [7].
7. DNA must be extracted from the blood immediately after its
collection.
8. Selection of the organs to be harvested depends on the goals of
the experiment. Tissues that are the main sites of disease
pathology, such as kidney, liver, and lung, are often collected.
However it has been shown that Leptospira can disseminate to
and colonize many other organs [11].
9. The exact medium composition and incubation scheme
depend on the conditions being examined. The number of
replicates to be tested depends on the conditions being examined and the number of mutants in the pool. A pilot experiment with a small number of mutants may be necessary to
calculate the appropriate number of replicates from a power
analysis.
10. Carefully remove the supernatant; pellets are loose due to the
low-speed centrifugation.
11. Calculate the volume of tissue to use for DNA extraction with
the following formula: volume in μl ¼ (500 μl  25 mg)/
(mg of diced tissue). For example, 250 μl homogenate will be
used from a 50 mg tissue resuspended in 500 μl PBS.
12. Wear earmuffs to protect hearing.
Transposon Sequencing in Leptospira interrogans
105