Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses

Safety Statement

This protocol describes infection of mice with live L. monocytogenes. The pathogen is handled safely under BSL2 conditions by trained personnel who are not immunocompromised. Immunocompromised people include pregnant women, the elderly and individuals who are HIV-infected or have chronic conditions that require treatment with immunosuppressive therapy. Personnel should don a protective lab coat or gown, gloves, mask, and eye protection while handling infected samples. The work described herein was performed under BSL2 conditions under a certificate (#32876) that was issued by the University Health Network (UHN) Biosafety office. Carcasses from infected mice or any unused tissues were double-bagged and disposed of in biohazard waste. Cages from infected mice were also decontaminated by autoclaving.

Ethics Statement

Mice were maintained and infected in a quarantine room within UHN animal facilities and were cared for in accordance with the guidelines set by the Canadian Council on Animal Care. All procedures on mice were carried out under animal use protocol #3214 that was approved by the UHN animal care committee. Due to ethical considerations, death was not used as an endpoint for survival studies. The modified LD₅₀ dose reported here for L. monocytogenes infection was determined to be the dose at which 50% of the mice reached specific endpoints, which consisted of a 20% loss in body weight or showing at least two of the following clinical signs: lethargy, ruffled fur, hunched posture, labored breathing, dull or sunken eyes. Mice were euthanized when they reached endpoints via exposure to carbon dioxide (CO₂) according to UHN facility guidelines.

1. Preparation of Glycerol Stocks for Long-term Storage

NOTE: This procedure describes how glycerol stocks of the EGD strain of L. monocytogenes are prepared from an original glycerol stock. Steps that have the potential to generate aerosols should be performed within a certified biosafety cabinet (BSC).

1. Prepare brain heart infusion (BHI) agar plates for bacterial growth. For this add 3.8% (w/v) BHI broth and 1.5% (w/v) agar to double distilled H₂O (ddH₂O). Autoclave liquid. Once the agar cools to 50 °C, dispense liquid into bacterial petri dishes (25 ml/dish) and let plates dry (uncovered) in the BSC for 1 hr.
   NOTE: Transfer BHI agar into a 50 °C water bath after autoclaving to avoid solidification prior to pouring plates. Store BHI plates at 4 °C upside down (with media side on top) until ready for use.
2. Prepare liquid BHI media. For this, mix 3.8% (w/v) BHI broth in ddH₂O. Autoclave.
3. Remove frozen glycerol stock of the L. monocytogenes EGD strain from the -80 °C freezer and thaw to room temperature.
4. Dip a sterile pipette tip in the thawed glycerol stock and immediately streak the tip back and forth across a section of a BHI plate. This is the primary streak.
5. Turn the plate by 90 °C and using a fresh pipette tip, drag through the first streak and spread it to the next ¼ of the plate (this is the secondary streak). Repeat once more to make the tertiary streak.
6. Turn plate upside down and incubate at 37 °C overnight. Single uniform colonies should be obtained in the last set of streaks and visible between 16 and 24 hr.
7. Dispense 10 ml of sterile BHI broth into a sterile vented 50 ml tube. Pick one colony of L. monocytogenes from the plate using a sterile pipette tip and inoculate the broth. Incubate the culture in a 37 °C orbital shaking incubator overnight or until OD₆₀₀ = 1.0 with settings at 225 rotations per min (rpm).
   NOTE: Glass or disposable plastic Erlenmeyer flasks can also be used to culture bacteria. Regardless of the type of container used, make sure that it is sterile, vented and that the volume of culture does not exceed 20% of the total volume of the container to ensure appropriate aeration of the bacteria.
8. Prepare glycerol stocks by mixing sterile 100% glycerol with overnight bacterial liquid culture at a 1:1 ratio. Distribute the bacterial/glycerol mixture into 2 ml cryogenic vials (500 μl/vial) and transfer vials to -80 °C freezer for storage.
   NOTE: Bead stock methods can also be used in place of glycerol stocks to store bacteria. By this method, porous microbeads are inoculated with a pure culture of L. monocytogenes and are stored at -80 °C. Each bead can be used to inoculate a fresh culture as needed. See materials list for further information.

2. Determination of Growth Curve of L. monocytogenes in Day Culture

NOTE: This procedure describes how to generate the growth curve for L. monocytogenes that is used to estimate the colony forming units (CFU) for infection studies. All steps that have the potential to generate aerosols should be performed within a certified BSC.

1. Take 100 µl of overnight culture generated in Step 1.7 to 10 ml BHI media in a vented 50 ml tube and grow at 37 °C in a shaking incubator (225 rpm, tilted at 45° angle). Use a non-inoculated tube as a control.
2. Take 0.5 ml samples of the culture at hourly intervals (1, 2, 3, 4, 5, 6 hr, etc.). Dilute each aliquot 1:1 (v/v) with BHI media in a plastic cuvette. Pipette up and down to mix. Measure the optical density (OD) at 600 nm (OD₆₀₀) using a spectrometer. Continue culturing bacteria until OD₆₀₀=1.
3. At the same time, take a 100 μl sample of the culture and dilute with 900 μl BHI media in a sterile 1.5 ml microcentrifuge tube (this is the 10^-1 dilution). Centrifuge the bacteria at 6,000 x g for 5 min, and aspirate the supernatant.
4. Wash bacteria twice by resuspending the pellet in 1 ml of BHI media, centrifuging for 5 min at 6,000 x g, and then aspirating the supernatant. Resuspend the pellet in 1 ml BHI media. Prepare a 10-fold dilution series of this sample in BHI media (10^-2 to 10^-9). Spread 100 μl of each diluent onto separate BHI agar plates. Incubate plates overnight at 37 °C in an incubator.
5. The next day, pick plates that have between 30 – 300 colonies. Discard the rest. Count the colonies on these plates. Table 1 shows an example of counts obtained in an aliquot that was taken when OD₆₀₀ = 0.84. In this example, one of the plates (i.e., 10^-6 dilution) had colony counts between 30 – 300 and was used for the CFU/ml calculation.
   NOTE: Plates with greater than 300 colonies are not used, since overcrowding can hinder bacterial growth and also makes it difficult to discern and enumerate individual colonies. Plates with counts < 30 are also not used because small errors in dilution technique or the presence of contaminants can have a large impact on the precision of counts at the lower end of the range.
6. Divide the number of colonies by the volume plated and then multiply by the dilution factor to obtain the CFU/ml value for a particular dilution. In the example in Table 1, the count at the 10^-6 dilution was 70. Divide this value by 0.1 ml to get the CFU/ml value for the diluted culture. Then multiply this value by the dilution factor (10⁶) to obtain the CFU/ml value of the undiluted culture (7.0 x 10⁸).
7. Plot the OD₆₀₀ (y-axis) versus time in h (x-axis) to identify the logarithmic phase of growth²⁶.
   NOTE: This growth curve provides an estimate of the CFU/ml of the day culture when grown to a certain OD reading. Choose a OD₆₀₀ reading that is in the logarithmic phase of growth that can be used as a target OD₆₀₀ for growing day cultures. These data now can be used to estimate the CFU in a culture for preparation of inoculum (Procedure 3).

3. Preparation of the Inoculum for Experimental Infection with L. monocytogenes

NOTE: This procedure describes the preparation of the infectious inoculum from a day culture that was started from an overnight culture (prepared in Procedure 2). All of these steps are performed in the BSC unless otherwise indicated.

1. Calculate the number of CFU required for infection based on the number of mice and experimental design of the study. Add an appropriate volume of BHI media to a sterile vented Erlenmeyer flask or culture tube.
   NOTE: The CFU of bacteria prepared will be dependent on the type of experiment performed. For studying NK and NKT cell responses during infection, each mouse is inoculated with 10⁵ CFU of bacteria (Procedure 6). If studying adaptive T cell responses to infection or measuring bacterial load, each mouse is inoculated with 2 x 10⁴ CFU of bacteria (Procedure 8). If studying survival to endpoints, each mouse is inoculated with the LD₅₀ dose of the pathogen (which is 10⁵ CFU for males and 1.5 x 10⁵ CFU for females, see Procedure 9).
2. Inoculate the tube containing BHI media with 100 μl of overnight culture. Incubate the culture in a 37 °C orbital shaking incubator (225 rpm) until target OD₆₀₀ is reached. Transfer culture contents into a sterile centrifuge tube.
3. Centrifuge bacteria into a pellet for 5 min at 6,000 x g using a centrifuge. Aspirate the supernatant using a vacuum attached to a trap flask containing bleach.
4. Wash pellet twice with 1x phosphate buffered saline (PBS), centrifuging (5 min at 6,000 x g) in between.
5. Aspirate the second wash and dilute bacteria at the appropriate concentration in 1x PBS to deliver the CFU of interest to each mouse in a 200 µl volume.
   NOTE: It is best to use a commercial source of sterile 1x PBS for washing bacteria and for preparation of the inoculum, since lab glassware can introduce immunological contaminants such as lipopolysaccharide.

4. Experimental Infection of Mice with L. monocytogenes

NOTE: This procedure describes how to infect mice with the inoculum prepared in Procedure 3 and how to verify the CFU delivered in the inoculum. Handling of mice and injections are performed in a BSC.

1. Order a sufficient number of male or female C57BL/6J mice for your experiment. Also order mice to serve as uninfected controls.
2. Allow mice to acclimatize for 1 week prior to bacterial inoculation.
   NOTE: This is because the stress associated with transport of the animals can trigger a transient increase in stress hormone production and lymphopenia^27,28.
3. On the day of inoculation, obtain a baseline body weight for each mouse and record it in the lab notebook.
4. In the BSC, mix the bacterial suspension up and down using a sterile pipette to ensure that the bacteria are evenly distributed and then take up 200 µl of the inoculum into a 1 ml safety engineered syringe fitted with a 25 G needle.
5. Inject a mouse i.p. with 200 μl of prepared inoculum (e.g., 10⁵ CFU for NK cell infection, Procedure 6). For this procedure, scruff mice with the less dominant hand by grabbing the loose skin around the mouse's shoulders. After ensuring that the mouse is well-restrained, inject the mouse in the lower quadrant of the abdomen, just lateral to the midline to avoid the bladder.
6. Dispose of the needle and syringe in a biohazard sharps container.
7. Repeat steps 4.3 – 4.6 until all mice are injected. Conduct similar steps with 1x PBS injected mice (non-infected controls).
   NOTE: Since the CFU is an estimate based on the growth curve, it is also good practice to check the actual CFU in the inoculum. For this, prepare 3 – 4 different dilutions of the prepared inoculum (using 10-fold dilution series) that you expect will result in countable colonies. Spread 100 μl of each diluent onto a BHI agar plate and incubate overnight at 37 °C. Count the colonies and calculate the actual CFU/ml as described in Procedure 2.

5. Preparing Heat-killed L. monocytogenes for Immune Studies

NOTE: All steps that have the potential to generate aerosols are performed within the BSC.

1. Grow day culture until OD₆₀₀ values are reached that are within the logarithmic phase. Dispense culture into 1.5 ml microcentrifuge tubes.
2. Incubate tubes in a 70 °C in a water bath for 1 hr to kill bacteria.
3. Wash bacteria twice with 1x PBS as in Steps 3.3 and 3.4. Resuspend in sterile complete RPMI media containing fetal calf serum (FCS) (see Supplemental File 1 for recipe) at a concentration of 4 x 10⁶/ml. Aliquot killed bacteria into 2 ml sterile cryogenic vials and store at -80 °C.
4. Confirm the death of the bacteria by spreading 100 μl of heat-killed bacteria preparation onto BHI agar plates and incubating overnight at 37 °C.
   NOTE: These heat-killed bacteria should be ready for stimulating lymphocytes in culture in Procedure 8. If there are any colonies growing on the BHI agar plate, repeat heat-killing procedure.

6. Measurement of IFN-γ Responses by NK and NKT Cells during Infection

NOTE: This procedure describes how to measure the IFN-γ responses by NK and NKT cells in mice at 24 hr after infection with 10⁵ CFU of the L. monocytogenes. This dose is used because it induces robust IFN-γ responses by NK and NKT cells in the spleen²⁴. Conduct all steps in the BSC. To help maintain cell viability, keep cells on ice whenever possible and use ice-cold buffers.

1. Inoculate mice as described in Procedure 4 with 10⁵ CFU of the L. monocytogenes. At the same time, inject non-infected control mice i.p. with an equal volume of 1x PBS.
2. Euthanize mice at 24 hr post-inoculation by CO₂ inhalation according to institutional guidelines.
3. Lie each mouse on its right side and wet down the skin with 70% ethanol using a squeeze bottle.
4. Using aseptic or sterile forceps and tough-cut scissors, incise the skin just below the bottom of the rib cage.
5. Spray down the exposed muscle layer with 70% ethanol. The spleen should be visible underneath the muscle layer (open arrow head in Figure 1).
6. Using aseptic or sterile forceps and fine scissors, incise the muscle layer to reveal the spleen. Gently grab the spleen with the forceps and use fine scissors to cut the spleen away from surrounding connective tissue.
7. Place the spleen in a 15 ml conical tube containing sterile 1x PBS.
8. Half-fill sterile petri dishes with sterile 1x PBS. Dissociate the spleen through a 70 μm nylon cell strainer into the petri dish using the flat end of a sterile 3 ml syringe.
9. Transfer the splenocyte suspension into a clean sterile 15 ml conical tube using a sterile 10 ml serological pipette.
10. Centrifuge samples at 335 x g for 10 min at 4 °C.
11. Aspirate the supernatant into a trap flask containing bleach. Loosen the cell pellet by flicking the tube with a finger or by dragging the bottom tube back and forth along a corrugated surface (e.g., air flow vent in the BSC).
12. Lyse red blood cells by adding 1.5 ml of Ammonium-Chloride-Potassium (ACK) lysis buffer (see Supplemental File 1 for recipe) to each spleen. After exactly 1 min and 15 seconds, fill the tube with 1x PBS to stop the cell lysis.
13. Centrifuge cells as described in step 6.10. Aspirate the supernatant and resuspend the cell pellet in 10 ml of fluorescence-activated cell sorting (FACS) Buffer (sterile 1x PBS containing 2% FCS).
14. Count the cells using a hemocytometer. For this, take two aliquots of cell suspension for counting. Add 15 µl of each cell suspension to an equal volume of Trypan blue (0.04%, made by diluting 0.4% trypan blue solution in ddH₂O). Load 15 µl of each cell/Trypan blue suspension into the chamber of the hemocytometer (i.e., one in the top chamber, one in the bottom chamber).
15. Using a microscope, count all non-blue cells in five large squares of the central grid in each chamber (Figure 2). Take this count and divide it by 10 to obtain the number of cells in 10⁶/ml. In the example in Figure 2, the count is 215 cells in the 5 squares; therefore, the cell concentration is 21.5 x 10⁶/ml. Average the cell concentrations obtained from the two samples.
16. Seed 1 x 10⁶ cells per well/stain in a 96-well round-bottom plate for flow cytometry staining. Make sure to also seed cells for unstained and fluorescence minus one (FMO) controls. See recommended flow cytometry staining panel in Table 2.
    NOTE: For the following steps, it is recommended to keep cells on ice or at 4 °C and to protect cells from light with foil when fluorochromes are present. A multichannel pipette can be used to dispense liquids into 96-well staining plates to speed up processing. Be careful not to disturb the cell pellet when aspirating the supernatant from the centrifuged plate. Staining can also be done in FACS tubes if the centrifuge is not fitted with plate adapters. All centrifuge steps from this point on are done at 456 x g for 5 min at 4 °C.
17. Centrifuge the plate and then wash cells twice with FACS buffer. One wash is done by adding 200 μl of FACS buffer to each well, centrifuging the plate, and then aspirating the supernatant.
18. Perform blocking step by adding 50 μl/well of FACS buffer containing anti-mouse CD16/CD32 (purified Fc block) (0.5 µg). Incubate cells at 4 °C for 15 min. Wash cells once in 1x PBS as described above.
19. Add 100 μl viability dye (fixable viability dye diluted 1:1,000 in 1x PBS) to cells. Stain cells at 4 °C in the dark (in refrigerator) for 30 min. Wash cells twice in FACS buffer as described above.
20. After a second wash, add 100 μl of cell surface antibodies or tetramers to respective wells according to the staining panel described in Table 2. Stain cells at 4 °C in the dark (in refrigerator) for 30 min. At this time, also add antibodies for staining single positive and FMO controls.
    NOTE: Regarding single positive controls, it is recommended to either use splenocytes that are stained with various fluorochrome versions of the CD4 antibody or commercial compensation beads that are stained with the antibodies used in the panel. Prior to conducting this staining procedure, all FACS antibodies should be titrated in test studies to determine optimal concentrations for staining.
21. Wash cells twice in FACS buffer as described above and then fix cells by resuspending them in 50 μl of 4% paraformaldehyde (16% paraformaldehyde stock diluted in ddH₂O) and incubating for 10 min at room temperature. CAUTION: The paraformaldehyde is toxic and should only be handled in the fume hood.
22. Wash cells twice in FACS buffer, centrifuging in between. Resuspend cells in FACS buffer. Continue to next step or store cells in the refrigerator protected from light for up to three days.
23. Centrifuge cells, remove the supernatant, and wash cells twice with 150 μl of 1x Permeabilization/Wash Buffer (Perm/Wash buffer), centrifuging in between. The Perm/Wash Buffer is prepared from a 10x stock by diluting 1:9 (v/v) in ddH₂O.
24. After the second wash, resuspend the cells in 150 μl of Perm/Wash Buffer and incubate for 15 min at 4 °C in the dark.
25. Centrifuge cells again and then aspirate the supernatant. Resuspend cells in 50 μl of 1x Perm/Wash buffer containing anti-IFN-γ and incubate for 1 hr at 4 °C in the dark.
26. Wash cells twice with 1x Perm/Wash buffer, centrifuging in between. Resuspend cells in 250 µl of FACS buffer and then transfer cells to FACS tubes.
27. Proceed to flow cytometry acquisition using a flow cytometer that has an appropriate laser configuration and filter set to discriminate fluorochromes used in the staining panel described in Table 2²⁹. Collect at least 200,000 events per sample and 10,000 events for compensation controls.
28. Apply compensation matrix and analyze data using flow cytometric analysis software³⁰.

7. Measurement of Bacterial Load in the Spleen and Liver at the Time of Peak Infection

NOTE: All steps are performed within a BSC unless otherwise noted.

1. Inoculate mice i.p. with 2 x 10⁴ CFU of the pathogen using procedures described in Procedure 4.
2. On day 3 post-infection, prepare sterile 1.5 ml microcentrifuge tubes, each containing 500 μl of ice-cold sterile 0.1% Triton X-100 in 1x PBS and 0.2 – 0.3 g of 1.5 – 2 mm acid-washed sterile glass beads. Weigh each tube.
   NOTE: Glass beads are acid washed by incubating in 10% acetic acid in a beaker on a magnetic stirrer for 1 hr. These beads are then extensively washed with ddH₂O to remove acid, are air-dried, and then autoclaved prior to use.
3. Euthanize mice by CO₂ exposure.
4. For dissection of organs, lay the animal on its back on a dissecting board and pin the limbs of the mouse to the board using 25 G needles. Disinfect skin by wetting with 70% ethanol.
5. Using sterile tough cut scissors, make a midline incision in the skin from the groin to the mid chest and then from the mid groin towards each knee and from the mid chest towards each elbow. Blunt dissect and reflect back the skin, pinning it open using 25 G needles.
6. Disinfect muscle layer by wetting it with 70% ethanol and then using sterile fine scissors make a midline incision in the peritoneal wall. Grab the xiphoid process with forceps. Then, using the same fine scissors, make cuts in the peritoneal wall from the xiphoid process laterally on each side, following the rib cage, just below the diaphragm to reveal the liver.
7. Cut out a ~100 mg piece of the liver (use the same lobe for all mice) using sterile scissors and place it in a pre-weighed 1.5 ml microcentrifuge tube.
8. Use forceps to gently push aside the organs on the left side of the peritoneal cavity to visualize the spleen. Gently grab the spleen with a pair of forceps and release it from the peritoneal cavity by cutting away the surrounding connective tissue.
9. Place the spleen in the pre-weighed 1.5 ml microcentrifuge tube containing beads. Transport tissues to the laboratory in a leak-proof container containing ice. Re-weigh the tubes containing the organs to determine the tissue weights in mg.
10. Homogenize the tissues by shaking the tubes using a bead mill homogenizer for 3 min at frequency of 30 Hertz.
    NOTE: The bead mill method is preferred for homogenization as it is amenable for processing a large number of samples and creates less mess and potential exposure to the pathogen. However, automatic homogenizers or autoclaved 2 ml manual glass tissue homogenizers could be used as an alternative.
11. Prepare a 10-fold dilution series of the homogenates in 0.1% Triton-X-100 in 1x PBS, ranging from (ranging from undiluted to 10^-7).
12. Spread 100 μl of each diluted homogenate onto a BHI agar plate (in duplicate) using a sterile spreader. Transfer plates to a 37 °C incubator and incubate overnight.
13. Keep the plates that contain between 30 and 300 colonies/plate, discard the rest. Count colonies on each plate and determine the mean number of colonies for duplicate spreads.
14. Calculate the CFU/mg according the following equation:
    CFU/mg = CFU/ml in the homogenate, multiplied by the ml of homogenate prepared, divided by the mg weight of the tissue homogenized.
    NOTE: For example, if a mean of 30 colonies were counted after plating 100 μl of 10^-2 diluted homogenate prepared from a 120 mg piece of liver that was homogenized in 0.5 ml, the calculations would be as follows:
    CFU/ml = 30 colonies x 100 (dilution factor) / 0.1 ml (volume spread) = 30,000 CFU/ml.
    CFU/mg = 30,000 CFU/ml x 0.5 ml homogenate / 120 mg tissue = 125 CFU/mg.

8. Effects of L. monocytogenes on IFN-γ Responses by CD4⁺ and CD8⁺ Cells

NOTE: This procedure describes how to measure IFN-γ production by splenic CD4⁺ and CD8⁺ T effector cells harvested at the time of the peak of the adaptive immune response (~ 7 d post-infection) using two methods: (1) flow cytometry to measure IFN-γ by CD4⁺ and CD8⁺ cells by intracellular cytokine staining, and (2) ELISA to measure total IFN-γ levels produced by splenocytes (includes all T cells). Procedures are performed within the BSC.

1. Infect mice by injecting i.p. with 2 x 10⁴ CFU of the pathogen using procedures described in Procedure 4.
2. On day 7 post-infection, euthanize mice by CO₂ inhalation according to institutional guidelines.
3. Dissect the spleen (as described above) and place in a 15 ml conical tube containing sterile 1x PBS. Transport the tubes to the laboratory in a leak-proof container containing ice.
4. Process the spleens into a single cell suspension, lyse red blood cells as described in section 6, and then resuspend cells in complete RPMI media containing 10% FCS. Count cells using a hemocytometer.
5. Set up cultures for measurement of IFN-γ responses. For this dispense cells (4 x 10⁶ in 1 ml/well) into 24-well plates together with and equal number (4 x 10⁶ or 1 ml/well) of thawed heat-killed L. monocytogenes (prepared in Procedure 5). Transfer cells to a 37 °C incubator.
6. After 20 hr of incubation, add 0.66 μl/ml of protein transport inhibitor to wells and continue incubations.
7. Four hr later, transfer plate to BSC and collect 500 µl of culture supernatant and freeze (at -80 °C) for the later measurement of IFN-γ levels using a commercial enzyme-linked immunosorbent assay (ELISA) kit³¹. Then collect cells into a sterile 15 ml tube. Wash wells with 1x PBS and pool this wash together with collected cells.
8. Conduct cell-surface staining and intracellular staining for IFN-γ on CD4⁺ and CD8⁺ cells as described in Procedure 6 except use the staining panel described in Table 3. Proceed to flow cytometer acquisition (collecting 200,000 events/sample) and analyze data²⁹ using flow cytometry analysis software³⁰.

9. Measuring Mouse Survival to Endpoints after L. monocytogenes Infection

NOTE: This procedure describes the effect of an agent on mouse survival to endpoints post-infection with the modified LD₅₀ dose of the pathogen. All these procedures are conducted in the BSC in the animal facility.

1. Inject mice i.p. with the modified LD₅₀ dose of L. monocytogenes as described in Procedure 4. This was determined to be 10⁵ CFU (for male) or 1.5 x 10⁵ CFU (for female)³¹.
2. Follow mice twice daily for clinical signs and record these signs and animal body weights in a lab notebook. Euthanize mice if they show a 20% loss in body weight or two clinical signs of listeriosis (lethargy, ruffled fur, hunched posture, labored breathing, dull or sunken eyes).
3. After 14 days, euthanize surviving mice via CO₂ inhalation.
4. Prepare Kaplan-Meier plots of the data by plotting the percent survival of each group against time³².
   NOTE: If mice succumb to infection (meet endpoints described in Ethics Statement), this usually occurs by day 5 post-infection (Figure 7).