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Modeling Neisseria meningitidis Infection in Mice: Methods and Logistical Considerations for Nasal Colonization and Invasive Disease

  • Kay O. Johswich
  • Scott D. Gray-OwenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1969)

Abstract

The single greatest barrier to studying the lifestyle of Neisseria meningitidis stems from its exquisite adaptation to life in humans, a specialization which prevents it from infecting other animals. This barrier to modeling meningococcal infection has been overcome by the provision of factors that allow the meningococci to overcome one or more aspects of host restriction, including the use of mice expressing receptors that allow mucosal colonization and/or the inclusion of serum factors that facilitate meningococcal replication during disseminated meningococcal disease. Here we discuss these advances, consider variables that influence the outcome of infection, and detail the technical requirements to establish robust and reproducible nasal colonization or sepsis. Once established, these models can then be used to study the meningococcal lifestyle and the immune response during infection, and to facilitate development of novel drug or vaccine-based approaches to intervene in meningococcal carriage and disease.

Key words

Mouse model Intraperitoneal infection Sepsis Nasal infection Mucosal colonization Transgenic mice 

1 Introduction

Neisseria meningitidis is a resident of the healthy human nasopharyngeal mucosa, where it resides as a normal member of the commensal microbiome. However, in rare instances (typically ~1/100,000 in industrialized nations, but as high as 100/100,000 during epidemic outbreaks), the bacteria can cause a rapidly progressing disseminated disease manifesting as sepsis (meningococcemia) and/or meningitis. What triggers this transition from asymptomatic carriage to invasive disease remains largely unknown, although genetic deficiencies that interfere with the bactericidal activity of the serum complement cascade, concurrent respiratory infections, and low humidity do increase the risk [1]. The inability of N. meningitidis to infect animals other than humans has hampered understanding of its lifestyle within the mucosa and its interaction with the immune system, which may confer immunity or cause the devastating clinical manifestations associated with invasive meningococcal disease.

In vitro experimentation has begun to reveal virulence factors that contribute to infection and, by virtue of the fact that they specifically bind only the human forms of their targets, mediate this host restriction. Classical work found that the systemic administration of exogenous iron to mice increased their susceptibility to systemic infection by intraperitoneally administered N. meningitidis [2] and, subsequently, that the human blood-derived iron transport protein transferrin could itself support meningococcal growth in vivo [3]. This has since been explained by the discovery that the meningococci express a surface receptor capable of extracting iron from human (but not other) transferrin [4, 5], and human transferrin-expressing transgenic mice have been developed to avoid the requirement for administering an exogenous iron source [6, 7]. Paralleling this effect, the meningococci express several surface proteins that specifically bind to the human form of the complement cascade regulatory protein Factor H [8], and infant rats expressing a transgene encoding human factor H are more susceptible to meningococcal sepsis than are wild-type animals [9]. While these serum factors facilitate meningococcal replication in blood, they do not allow nasal colonization of the animals. Recently, we have observed that transgenic mice expressing human CEACAM1, which is targeted by the meningococcal Opa protein adhesins, is sufficient to allow prolonged mucosal colonization following nasal instillation [10, 11]. This model provides the first opportunity to study meningococcal adaptation, antibiotic susceptibility, and vaccine efficacy during this requisite early stage of infection.

Herein, we discuss logistical considerations and technical details to allow performance of both nasal colonization and invasive disease caused by N. meningitidis in mice.

2 Materials

2.1 General Materials and Devices

  1. 1.

    Biosafety cabinet.

     
  2. 2.

    Photometer.

     
  3. 3.

    Mouse weight scale.

     
  4. 4.

    Contact-free skin thermometer (infrared).

     
  5. 5.

    Infrared lamp.

     
  6. 6.

    Carbon dioxide supply with adjustable flow rate.

     
  7. 7.

    Alcoholic skin disinfectant.

     
  8. 8.

    Mouse preparation support pad (styrofoam).

     
  9. 9.

    Cotton swabs, single bud, autoclaved.

     
  10. 10.

    Columbia sheep blood agar plates (commercial).

     
  11. 11.

    Mouse restrainer (with opening for tail and opening for nose). Restrainers can be purchased from commercial vendors. Alternatively, they can be prepared from 50 mL conical tubes by cutting off the tip (hole for the nose) and drilling a hole into the lid (hole for the tail), respectively. Make sure there are no sharp plastic edges.

     
  12. 12.

    PBS (10×): Dissolve 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4 in 950 mL of distilled water and transfer to a graduated cylinder. Adjust pH to 7.4 with 25% hydrochloric acid (if pH is too high) or with 1 mol/L sodium hydroxide solution (if pH is too low). Add distilled water to make 1.0 L. For 1× PBS (“PBS”), mix 1 volume of 10× PBS with 9 volumes of distilled water.

     

2.2 Materials Specific for Intraperitoneal Infection

  1. 1.

    Brain-heart infusion (BHI) broth: Dissolve 37 g of brain heart infusion broth media in 1 L of distilled water. Autoclave at 121 °C for at least 15 min. Let cool down, make 50 mL aliquots under aseptic conditions and store at 4 °C until use. Let BHI warm to room temperature before use.

     
  2. 2.

    PBS + 5 U/mL heparin: To 5 mL of PBS, add 5 μL of 5000 U/mL unfractionated heparin solution and filter sterile. Store solution at 4 °C for up to 4 weeks.

     
  3. 3.
    Iron dextran solution (see Notes 1 and 2 ):
    1. (a)

      Mix 1 volume of iron dextran solution containing 100 mg/mL iron (commercially available, e.g., from Sigma Aldrich) with 9 volumes of sterile isotonic saline (endotoxin free, commercially available).

       
    2. (b)

      Sterilize solution using a syringe equipped with a 0.2 μm sterile filter.

       
    3. (c)

      Prepare enough for two injections per mouse, with each mouse receiving 200 μL per injection. Add 50% to the total calculated volume to compensate for the void volume of syringes used for injection.

       
    4. (d)

      Directly prior to infection, prepare one 1 mL syringe with a 26G 3/8″ cannula and, in a biosafety cabinet, fill it with 200 μL of the iron dextran solution for each mouse to be infected. Make sure that no air bubbles are in the syringe.

       
    5. (e)

      Store excess iron dextran solution at 4 °C, as it will be used 12 h after infection.

       
    6. (f)

      Plate out 50 μL of the prepared iron dextran solution on Columbia sheep blood agar plates and incubate for 2 days at 37 °C, 5% CO2 with saturated water atmosphere in order to verify sterility.

       
     

2.3 Materials Specific for Intranasal Infection

  1. 1.

    PBS + 1 mM MgCl2: To make a 1 M stock of MgCl2, dissolve 10.165 g MgCl2·6H2O in a total volume of 50 mL distilled water and then sterilize by autoclaving. Add 50 μL of 1 M stock of MgCl2 to 50 mL of sterile PBS to make PBS + 1 mM MgCl2.

     
  2. 2.

    Sterile polyester tipped applicators, 5½″, ultra fine aluminum handle (“applicator swab,” e.g., from Puritan) (see also Fig. 1).

     
  3. 3.

    1 mL syringe fitted with 20G 1″ needle and tube adaptor: Fit 20G 1″ cannula onto a 1 mL syringe. Equip the cannula with ~2.5 mm of fine bore polythene tubing (0.86 mm inner diameter, 1.27 mm outer diameter), cut the tube tip to a 45° angle (see also Fig. 1). First, draw 200 μL of air into the syringe, then 200 μL of PBS + 1 mM MgCl2.

     
  4. 4.
    Selective agar for recovery of N. meningitidis from mouse nasal samples: Selective agar plates (Thayer-Martin) are commercially available. Alternatively, they can be prepared as follows:
    1. (a)

      In a 1 L Erlenmeyer-flask, dissolve 36 g of GC medium base (e.g., from BD-Difco) to 500 mL of distilled water. Boil for 1 min to completely dissolve powder.

       
    2. (b)

      In a separate 1 L Erlenmeyer-flask, dissolve 10 g hemoglobin (e.g., from BD-Difco) in 500 mL of distilled water. Boil for 1 min to completely dissolve powder.

       
    3. (c)

      Autoclave both solutions at 121 °C for at least 15 min, let cool to ~50 °C and combine both solutions.

       
    4. (d)

      Add growth supplement for fastidious bacteria, which is commercially available (e.g., IsoVitalex from BBL or PolyVitex from Biomerieux), as per manufacturer’s specifications (see Note 3 ).

       
    5. (e)

      In order to suppress overgrowth of Neisseria by endogenous flora, add VCNT inhibitor (commercially available) as per manufacturer’s instructions (see Note 4 ).

       
    6. (f)

      Pour 15–18 mL of the liquid agar per 90 mm Petri-dish under aseptic conditions and let solidify. Store the plates in a sealed bag at 4 °C in the dark for up to 3 months.

       
    7. (g)

      Always let plates pre-warm to room temperature before inoculating with N. meningitidis.

       
     
Fig. 1

Special equipment required for sampling mouse nasal cavity. Top: Sterile polyester mini tip applicator with aluminum handle for swabbing the nasal cavity. Bottom: A 1 mL syringe equipped with a 20G 1″ cannula to which a tube adaptor (fine bore polythene tubing, 0.86 mm inner diameter, 1.27 mm outer diameter) is attached

3 Methods

3.1 General Remarks on Mouse Holding and Preparation

It is mandatory that approval by the appropriate ethics committee and governmental agency has been obtained before conducting any experiments using mice. The animals must be housed in a suitable facility according to all applicable regulations by local and federal guidelines and laws applicable to animal welfare, occupational health and safety and biotechnology. Mice should be free of specified pathogens (SPF) as listed by the Federation of Laboratory Animal Science Associations (FELASA) to avoid interference of other pathogens with the results of the experiments. Typically, mice are held at 22 °C with 55% ± 10% relative humidity with tenfold air exchange in holding rooms with a 12 h light/dark cycle. Animals are fed rodent standard diet and water as per convention of the respective animal facility. Infected animals must be held in aerosol-tight, individually ventilated cage (IVC) systems in order to prevent transmission of N. meningitidis to the handlers. If mice need to be transferred from a holding or breeding room to a specialized room for the infection, this should be done at least 5 days prior to infection in order to acclimatize to the new environment and, thereby, reducing the contribution of stress as a variable. If the design of the study mandates to obtain blood samples of the mice prior to infection, the acclimatization time should be extended appropriately to let the mice first acclimatize before the blood draw and recover for at least 5 days after the blood draw. The endemic mouse microbiome varies between facilities and, in fact, between cages. While published infectious doses may be used as guidelines for a particular strain, variation such as mouse microbiome, strain culture conditions and the inherent phase and antigenic variation of meningococcal strains means the infectious dose must be reestablished for each strain in each facility since the outcome may vary significantly.

3.2 Mouse Lines

For the intraperitoneal infection model, male mice aged 6–8 weeks are used, with body weights typically ranging between 22 g and 28 g. In principal, any inbred mouse line or mutant strain hereof (e.g., knockout strains) can be used; however, we typically use C57Bl/6J. The use of transgenic mice expressing human transferrin avoids the need to add an exogenous iron source to support the infection, while mice expressing other serum-derived factors that the meningococci bind (e.g., lactoferrin, factor H) may also facilitate meningococcal growth in vivo. For intranasal infection, human CEACAM1-expressing mice are required [11], and have been used successfully in both the FvB or C57Bl/6J genetic background (see Note 5 ). Thus far, we have not observed any influence of age or gender of the mice on the colonization frequency or amplitude [11]; however, caution should be taken when looking at inflammatory responses, as male mice may mount stronger responses than females [12].

3.3 The Mouse Intraperitoneal Infection Model

The protocol given here is a recommendation based on our experience, but it might be modified and further developed to accommodate differences in the mouse lines, bacterial strains and/or circumstances regarding local facilities and legal regulations. This protocol is intended for studies that monitor mouse clinical presentation and survival as primary readout and bacteremia as secondary readout.

3.3.1 Inoculum Preparation

All steps involving N. meningitidis liquid cultures must be performed in a biosafety cabinet. Unless stated otherwise, all steps are done at room temperature.
  1. 1.

    On the day before the infection, streak out N. meningitidis onto Columbia sheep blood agar plates and incubate at 37 °C, 5% CO2 with a humidified atmosphere for 20 h.

     
  2. 2.

    With a cotton swab, collect 20–30 colonies from overnight growth and spread them onto a fresh, pre-warmed Columbia sheep blood agar plate and incubate for 4 h at 37 °C, 5% CO2 with a humidified atmosphere to obtain a log-phase culture (see Note 6 ).

     
  3. 3.

    Harvest the entire log-phase culture with a cotton swab and resuspend it in 1 mL of BHI broth to obtain an N. meningitidis stock suspension.

     
  4. 4.

    Make a 1:20 dilution (50 μL stock suspension +950 μL BHI broth) and transfer 800 μL thereof to a half-micro cuvette to measure the optical density at 600 nm (OD600) against plain BHI as reference. An OD600 of 1.0 corresponds to approximately 1.5 × 109 colony forming units (CFU) per mL when working with strains MC58 or H44/76 (see Note 7 ).

     
  5. 5.

    Calculate optical densitiy of the stock suspension (based upon the CFU at the measured OD600 of a 1:20 dilution, multiplied by 20). To 1000 μL of BHI, add (1000/(OD600 - 1)) μL of the stock suspension to obtain and OD600 of 1.0, which corresponds to 1.5 × 109 CFU/mL. Transfer 800 μL of the stock suspension to half-micro cuvette to verify OD600. A range from ±5% is acceptable. As the cuvettes are usually not sterile, discard cuvette and content.

     
  6. 6.

    From the suspension with 1.5 × 109 CFU/mL, mix 30 µL with 870 μL of BHI to obtain 5 × 107 CFU/mL. Then, add 100 μL of the obtained dilution to 900 μL of BHI to obtain 5 × 106 CFU/mL. Finally, make another dilution to obtain the final inoculum (i.e., 1:10 to achieve 5 × 105 CFU/mL) (see Note 8 ). Per mouse, 200 μL of inoculum is used for intraperitoneal injection. To calculate the total volume of inoculum required, multiply 200 μL with the number of animals to be infected and add 50% volume to compensate for the void volume of syringes used for injection. Handle the inoculum at room temperature as this will keep the bacteria viable but does not allow for growth. However, the inoculum should be used within 1 h after preparation.

     
  7. 7.

    In order to verify the bacterial density in the inoculum, mix 20 μL of the inoculum (5 × 105 CFU /mL) with 980 μL of BHI (1:50 dilution). Then, make three serial 1:2 dilutions by adding 200 μL of the prior dilution to 200 μL BHI and pipetting to mix but not generate bubbles. Plate out 50 μL of each of these four dilutions on Columbia sheep blood agar plates, which should yield ~500, 250, 125, and 63 colonies, respectively, after overnight incubation at 37 °C, 5% CO2 in a humidified atmosphere. This step is done right after inoculum preparation and again after infection to monitor any changes in CFU/mL during the time period required for animal handling. These results will not be available until ~16 h after inoculation of the mice, but are helpful if the outcome of infection is unexpected.

     
  8. 8.

    Directly prior to infection, prepare one 1 mL syringe with a 26G 3/8″ cannula for each mouse and fill each with 200 μL of the inoculum. Make sure that no air bubbles are in the syringe.

     

3.3.2 Intraperitoneal Infection

  1. 1.

    The infection must be done in a biosafety cabinet in the handling room of the animal facility. Restrain the mouse tightly in supine position to have their abdomen exposed facing up.

     
  2. 2.

    Inject 200 μL of the iron dextran solution (see Subheading 2.2, item 3d) in the upper left quadrant of the mouse abdomen. Be careful to insert the needle deep enough to obtain intraperitoneal, rather than subcutaneous, application. Avoid puncturing the intestine or major blood vessels as this will lead to systemic infection by intestinal flora and/or bleeding that will make results unusable.

     
  3. 3.

    Inject 200 μL of the inoculum in the lower right quadrant of the mouse abdomen. Once again, be careful to insert the needle deeply enough to obtain intraperitoneal infection, and avoid puncturing the intestine or major blood vessels as this will lead to unusable results (see Note 9 ).

     
  4. 4.

    At 12 h after infection, inject a second bolus of 200 μL of iron dextran solution (see Subheading 2.2, item 3e) in the upper left quadrant of the mouse abdomen. This is necessary to provide enough iron for bacterial growth. Skipping this step will suppress the ongoing infection.

     

3.3.3 Mouse Monitoring

  1. 1.

    From 36 h before infection and then every 12 h until infection (0 h), monitor the health status, weight, and temperature of the mice. Use a mouse scale to weigh the mice. Use a contact-free infrared skin thermometer to measure dorsal temperature of mice, by scanning the mouse dorsally from nose to tail and note the highest temperature measured. We have found that some pediatric thermometers, which can be purchased from a pharmacy, can work well for this purpose if they work over the range of normal mouse body temperature (34–38 °C). Use a scoring scheme to reproducibly grade the mouse condition. Two examples for scoring schemes for mouse health monitoring are shown in Tables 1 and 2.

     
  2. 2.
    For ethical reasons, the animal welfare acts of most countries do not permit experiments in which vertebrates are left to die on their own, particularly as consequence of manipulations such as infections. Thus, humane endpoint criteria must be used to determine imminent death as early and as accurately as possible to avoid unnecessary suffering while allowing accurate prediction of the outcome. The humane endpoint may be determined by a combination of clinical manifestations leading to a set clinical score, depending on the scoring system used. However, when animals reach any one of the following humane endpoint criteria, they must be immediately sacrificed. Humane endpoint criteria for this model are:
    1. (a)

      shivering or tremors;

       
    2. (b)

      problems immediately rising from a lateral position; or

       
    3. (c)

      body temperature below 34 °C (as measured dorsally using an infrared thermometer) at three consecutive scoring timepoints.

       
     
  3. 3.

    Monitoring of the mice after infection must be done with the intervals specified in the protocols approved by the local Ethics committee and the appropriate governmental agencies. Typically, we routinely check on the infected animals at 3 h post-infection to make sure they are healthy (which may not be the case if, for example, the intestine is punctured during intraperitoneal injection). First symptoms and signs of distress become visible after 9 h of infection and are clearly apparent at 12 h. Symptoms aggravate further and peak around 24 h, at which point non-survivors usually reach the humane endpoint criterion. Generally, monitoring should be done every 6 h after infection. However, as non-survivors reach the humane endpoint around 24 h, it is advised to increase the monitoring frequency as per the actual condition of the mice between 12 h and 36 h for timely detection of the humane endpoint. After 48 h, the survivors start to recover and the scoring frequency might be lowered commensurate with the actual status of the mice. Recovery of the mice is usually complete within 7 days after infection in severe cases.

     
  4. 4.

    If an animal reaches the humane end point, it must be immediately euthanized according to the protocols approved by the local Ethics committee and appropriate governmental agencies. CO2 asphyxiation is recommended to euthanize the mice (see Note 10 ).

     
  5. 5.

    To sacrifice a mouse, place it in a chamber with a lid equipped with a connection to an adjustable CO2 supply. The lid should not be too tight since it must allow for escape of the air, which is gradually displaced by the heavier CO2 accumulating at the bottom. Adjust the flow rate to obtain 50–100% chamber replacement rate (i.e., for a 2 L chamber, set CO2 flow rate to 1–2 L/min). Do not flood chamber with CO2 before placing the mice in, as this is noxious to the animals [13]. Wait until mouse stops breathing for at least 30 s before removal from the chamber. If required, cardiac puncture can be performed after this point to obtain a blood sample.

     
Table 1

Example 1 for clinical scoring scheme

Symptoms

Score

Unprovoked behavior

Normal

0

Inactive, decreased alertness

1

No unprovoked mobility, self-isolation

2

Shivering, tremors

3

Provoked behavior

Normal

0

Slightly abnormal reaction

1

Slow reaction, reduced grasping force

2

Problems to rise from lateral position

3

Feces

Normal

0

Diarrhea, soiled perianal region

1

Blood in stool, severe diarrhea

2

Body weight changes

0–5%

0

>5–10%

1

>10–15%

2

>15%

3

Body temperature

≥35.4 °C

0

35.0–35.3 °C

1

34.0–34.9 °C

2

≤34.0 °C

3

Grooming

Normal

0

Moderate lack of grooming (dull coat)

1

Severe lack of grooming (rough coat, dirty appearance)

2

Posture

Normal

0

Slightly abnormal

1

Severely abnormal, strongly hunched

2

Eyes and nose

Normal

0

Exudate visible

1

Eyes closed and exudate, sunken eyes

2

Breathing

Normal

0

Slightly abnormal

1

Severely abnormal, exerted breathing

2

Table 2

Example 2 for clinical scoring scheme

Symptoms

Score

Body weight

Increased or <3% loss

0

>3–5% loss

1

>5–10% loss

5

>10–20% loss

10

>20% loss

20

General condition

Normal (smooth, shiny coat, normal posture, clear eyes)

0

Small deviations (e.g., coat slightly dull)

1

Dull coat, piloerection, slightly hunched position

5

No grooming, soiled perianal region, eyes partly closed or with exudate, hunched position

10

Tremors/shivering, very strong dehydration, problems rising from lateral position

20

Behavior

Normal (agile, curious)

0

Small deviations

1

Reduced mobility

5

Self-isolation, lethargy, coordinative deficits, reduced defensive behavior

10

Very weak grasping force, very weak/no defensive behavior

20

Clinical

Normal (breathing frequency, mouse feels warm)

0

Small deviations

1

Breathing frequency ± 30%

10

Exerted breathing, mouse feels cold

20

3.3.4 Taking Samples

  1. 1.

    At 3 h after infection, a tail vein blood sample of each mouse is taken to monitor bacteremia. Place mouse in a restrainer and use skin disinfectant to disinfect the tail. Warm the tail under infrared lamp for 10 s to dilate the tail veins.

     
  2. 2.

    Carefully puncture the tail vein with a 25G ½″ needle and carefully stroke over the tail from rostral to caudal to expel a droplet of blood. Take up 5 μL of blood using pipet with a sterile tip and dilute this in 45 μL of PBS + 5 U/mL heparin, laid in the top row of 96-well plates (see Note 11 ). Disinfect the tail after the procedure with skin disinfectant.

     
  3. 3.

    When all blood samples are collected, make tenfold serial dilutions by transferring 10 μL of the diluted blood to 90 μL of plain PBS laid in a row on the same 96-well plate to obtain a total of 6 dilutions of each sample. Plate out 20 μL of each dilution on Columbia sheep blood agar plates using a pipettor (or a multichannel pipettor if available) by carefully dropping the 6 dilutions of each sample in a line on a single agar plate and then tilting the plate to a 60° angle to let the droplets run down to the plate surface. Make sure that the droplets yield uniform lanes and do not mix. Reverse the plate angle shortly before the droplets touch the rim of the plate and then let the plate sit leveled until liquid has been absorbed by the agar.

     
  4. 4.

    To monitor the course of bacteremia over time, take blood samples at 12 h, 24 h, 36 h, 48 h and then every 24 h (see Note 12 ). Additionally, take tail vein blood samples of mice when they reach the humane end point. It is recommended to also take cardiac blood from the sacrificed mice and save for later (e.g., cytokine or antibody) analyses and/or plate these as well (see Note 13 ). It is important that a maximum of 200 μL blood can be sampled from an individual mouse over a 2 week period, so volumes obtained must be carefully monitored.

     
  5. 5.

    Enumerate colonies after incubation overnight at 37 °C, 5% CO2 with a humidified atmosphere. Calculate the number of CFU/mL blood by multiplying the counted colonies with the dilution factor (which is 10 in the least-diluted lane, as 5 μL blood were diluted in 45 μL PBS + 5 U/mL heparin, and then multiply this by 50, since 20 μL sample were plated per lane).

     
  6. 6.

    (optional) If required, additional blood sample can be taken for measurement of plasma factors or for differential blood counts. However, as noted above, the amount of blood taken should be minimal in order to avoid unnecessary stress for the mice. For the analysis of cytokines or complement activation products, it is recommended to draw 10 μL of blood at 12 h of infection, where all mice are still alive and show clear signs of disease, including a robust cytokine response. Immediately dilute the blood in 90 μL of ice-cold PBS + 10 mM EDTA and store on ice before further processing. Supernatant is recovered after centrifugation at 10,000 × g for 5 min and 4 °C and frozen at −80 °C until analysis (e.g., by ELISA).

     

3.4 The Mouse Intranasal Infection Model

The protocol given here intends to monitor N. meningitidis nasal colonization, reflected as the number of N. meningitidis that can be recovered from nasal tissues after infection. From our experience, wild-type mice are not susceptible to N. meningitidis nasal infection unless the mice express human CEACAM1 [11]. The protocol is a recommendation based on our experiences, but it might be modified and further developed to accommodate different mouse lines, bacterial strains and/or circumstances in the local facilities and legal regulations.

3.4.1 Inoculum Preparation

  1. 1.

    All steps involving N. meningitidis liquid cultures must be performed in a biosafety cabinet; unless stated otherwise, all steps are done at room temperature (see Notes 14 and 15 ).

     
  2. 2.

    Follow the same steps as described in Subheading 3.3.1 to obtain a suspension with OD600 of 1.0, BUT replace BHI with PBS + 1 mM MgCl2 in all steps (see Note 16 ).

     
  3. 3.

    Of the suspension with 1.5 × 109 CFU /mL, mix 30 µL with 420 μL PBS + 1 mM MgCl2 (1:15 dilution; 1 × 108 CFU/mL). Subsequently, add 1 volume of the resultant suspension to 9 volumes of PBS + 1 mM MgCl2 to obtain the inoculum of 107 CFU/mL (this will provide 105 per mouse; adjust to achieve targeted inoculum). To calculate the total volume required, multiply the number of animals to be infected with that strain by 10 μL per infection and add 100 μL for safety. Do not administer more than 10 μL per mouse (~5 μL per nostril) since this may allow the inoculum to flow into the lung.

     
  4. 4.

    Verify the inoculum by making three serial 1:10 dilutions by transferring 40 μL of the prior suspension to 360 μL PBS + 1 mM MgCl2. Once the last dilution is completed, mix 200 μL of each with 200 μL PBS + 1 mM MgCl2 and then repeat this to obtain three serial 1:2 dilutions. Plate out each 50 μL of the last 4 dilutions onto Columbia blood agar plate and incubate at overnight at 37 °C, 5% CO2 in a humidified atmosphere. Enumerate grown colonies. Expected are ~500, 250, 125, and 63 colonies on the respective plates.

     

3.4.2 Intranasal Infection

  1. 1.

    The infection must be done in a biosafety cabinet in the handling room of the animal facility. It is advised that an additional disposable laboratory coat with plastic impregnation is worn on top of the regular gown; also, the gloves should be taped to the sleeves of the gown and an additional pair of gloves should be donned to allow for easy and safe glove exchange. A face mask must be worn (see Note 17 ).

     
  2. 2.

    Restrain the CEACAM1-humanized mouse in a restrainer allowing for access to the mouse nose and hold the restrained mouse in supine position (Fig. 2). Wait for the mouse to adjust to the situation (see Note 18 ).

     
  3. 3.

    Slowly pipet 10 μL of the inoculum directly onto the nares of the mouse, alternating between nares for each droplet that appears during pipetting, and let the mouse aspirate these (Fig. 2). When the inoculum is applied, place the mouse back in its cage.

     
Fig. 2

Intranasal application of N. meningitidis inoculum to the mouse. Alert mouse is placed in a restrainer (50 mL Falcon tube with tip cut off; round any sharp edges before placing animal inside) and held in supine position. When the mouse has adjusted to the restraining, carefully apply the inoculum (10 μL) dropwise to both nares

3.4.3 Monitoring the Mice

  1. 1.

    Beginning 2 days prior to infection, weigh the CEACAM1-humanized mice once per day, take their temperature and apply an appropriate scoring scheme as approved by the appropriate ethics committee and the governmental agencies. For two sample scoring schemes, see Tables 1 and 2.

     
  2. 2.

    In our experience, intranasal infection of CEACAM1-humanized mice does not lead to any visible signs of discomfort or invasive disease, particularly when no suitable source of iron to support meningococcal replication in the blood is administered. However, the actual health status of the mice must be assessed once every day until the end of the experiment.

     

3.4.4 Taking Samples

  1. 1.

    After 3 h, a tail vein sample should be obtained and plated as described under Subheading 3.3.4 to monitor for potential (unexpected) bacteremia.

     
  2. 2.

    CEACAM1-humanized mice can harbor N. meningitidis for as long as 14 days in their nasal tissues; we often use a day 3 endpoint to monitor the level of infection, either when comparing strains or the effect of vaccine, since this provides a very clear distinction between mice that become colonized and those that do not. Note that sampling at day 1 will rarely reveal some bacteria in WT (non-CEACAM1) mice, but these are cleared shortly thereafter.

     
  3. 3.

    Since the bacteria access the airways, bacterial detection must be done at the experimental endpoint. To harvest viable N. meningitidis from the mouse nasopharynx, sacrifice the mice by CO2 asphyxiation and exsanguinate by cardiac puncture (see Note 19 ). Fix the mouse in supine position on a styrofoam pad using preparation needles or cannulae.

     
  4. 4.

    With scissors and forceps, carefully expose the trachea and make a small incision 3 mm rostral of the clavicle (Fig. 3a). Using the prepared 1 mL syringe with a 20 G 1″ needle fitted with a tube adaptor filled with 200 μL PBS + 1 mM MgCl2 (see Fig. 1), carefully insert the needle into the incision and slowly flush the upper airways (see Note 20 ). Collect the lavage fluid in a 1.5 mL tube placed over mouth and nose of the mouse (Fig. 3b).

     
  5. 5.

    Next, cut off the nose tip, removing as much cartilage as possible. Make two incisions along the premaxilla to expose the nasal airways (Fig. 3c). Fix the skull pointing upward using forceps and insert a PBS-wetted aluminum applicator into the opened airways (Fig. 3d). This step requires practice, as enough pressure must be applied to insert the applicator deep enough to harvest the mucosa, but the applicator must not be bent. Thoroughly move the applicator up and down until the mucosa is well homogenized and sticking to the applicator. Remove the applicator and resuspend tissue debris into 500 μL (‘nasal swab sample’). Repeat this step 2 times to harvest as much nasal tissue as possible. Probe the nasal cavity a last time with the applicator and spread out the adherent tissue debris directly onto Thayer-Martin-Agar to quantify CFUs from this (‘swab direct’).

     
  6. 6.

    Plate out up to 200 μL of nasal wash onto a single Thayer-Martin agar plate. Plate out the “nasal swab sample” onto Thayer-Martin agar plates, with the entire sample distributed onto two individual agar plates. Usually, the samples do not need to be further diluted, as the number of recovered colonies usually ranges below 1000 in total (i.e., distributed over four plates); this low number may reflect the fact that a small tissue piece with many bacteria attached will generate a single colony. Together with the ‘swab direct’ sample, incubate all agar plates overnight at 37 °C, 5% CO2 in a humidified atmosphere.

     
  7. 7.

    Enumerate colonies and plot sum from all plates for each individual mouse as number of recovered N. meningitidis.

     
Fig. 3

Sample preparation after intranasal infection. (a) Fix the euthanized mouse with needles to a styrofoam support and remove fur from the neck region. Expose the trachea and make a small incision as indicated. (b) Place 1.5 mL tube over the mouse nose and fix with a needle. Insert tubing adaptor of the 1 mL syringe with 20G 1″ cannula and carefully flush the nasal airways. The lavage fluid is collected in the 1.5 mL tube at the mouse nose. (c) Cut off the nose tip and make incisions in the premaxilla as indicated. Carefully open the nasal cavity using a syringe. (d) Fix the mouse head tightly with forceps and insert a pre-wetted applicator swab to thoroughly swab the nasal cavity. Collect the material stuck to the applicator swab by thoroughly rubbing it to the walls of a 1.5 mL tube containing 500 μL PBS + 1 mM MgCl2

3.5 Data Analysis

  1. 1.

    In order to compare the significance of mouse survival rates among different groups at the end of the experiment, Fisher’s exact test can be used for statistical analysis. In order to account for the time course of deaths occurring in each group, the Mantel-Cox test or Kaplan-Meier test is suitable.

     
  2. 2.

    In order to analyze differences in the clinical scores of different mouse cohorts, non-parametric tests are mandatory. Two groups may be compared using the Mann-Whitney test, whereas comparison of three or more groups requires ANOVA on the ranks, such as the Kruskal-Wallis test.

     
  3. 3.

    In order to analyze differences in the bacteremia of mouse cohorts, or the number of bacteria recovered from the nasopharynx, the data distribution must be analyzed first in order to conduct the right statistical test. The D’Agostino-Pearson test or Kolmogorov-Smirnoff test is suitable to assess whether the data points follow normal (Gaussian) distribution. If the data are normally distributed, a Student’s T-test can be used to compare two groups, whereas a one-way-ANOVA is required for three or more groups. If the data are not normally distributed (which often occurs), a non-parametric test such as Mann-Whitney (2 groups) or Kruskal-Wallis-test (>2 groups) is used. Alternatively, the data can be log-transformed and tested again for normality. When doing so, it is imperative to clearly indicate the log-transformation, such as by clear labeling on the y-axis of a plot showing the data.

     

4 Notes

  1. 1.

    Instead of iron dextran, human transferrin can be used to provide a source of iron for N. meningitidis growth. Make a solution of 40 mg/mL human holo-transferrin (e.g., from Sigma Aldrich) in BHI and filter sterilize it. Per infection, mix 200 μL of transferrin solution with 200 μL inoculum and apply intraperitoneally as one single injection of 400 μL. If a second infection is to be performed in an animal, only one of the infections can involve human transferrin as a rapidly progressing allergic reaction occurs upon second exposure to the foreign transferrin. Thus, in this circumstance one of the infections should instead use iron dextran as an iron source.

     
  2. 2.

    Iron supplementation is not required when infecting transgenic mice which express human transferrin.

     
  3. 3.

    Instead of commercial growth supplement, Kellogg’s supplement can be used at 10 mL per 1 L of Thayer-Martin agar. To prepare Kellogg’s supplement, dissolve 40 g d-glucose, 1 g l-glutamine, 2 mg thiamine pyrophosphate, and 5 mg iron(III)nitrate in distilled water to yield 100 mL final volume. Filter sterilize this and make 10 mL aliquots of Kellogg’s supplement and freeze these at −20 °C until use.

     
  4. 4.

    As an alternative to commercial VCNT inhibitor, the antibiotic cocktail suppressing endogenous flora can also be prepared by reconstitution of 3 mg vancomycin, 7.5 mg colistin, 12,500 units nystatin, and 5 mg trimethoprim lactate in 10 mL water, which will suffice for 1 L of Thayer-Martin agar.

     
  5. 5.

    FvB mice are genetically deficient in components of the hemolytic complement cascade, as are some other mouse lines (e.g. DBA/2J, A/HeJ, A/J, AKR/, NZB/B1NJ, SWR/J, B10.D2/oSnJ).

     
  6. 6.

    Optionally, the bacteria can be subjected to iron starvation, in order to upregulate virulence factors [14]. Use a cotton swab to harvest the lawn of overnight growth on Columbia sheep blood agar plates and resuspend this in 10 mL BHI containing 60 μg/mL of the iron chelator desferoxamine mesylate. Incubate at 37 °C in a 50 mL conical tube for 4 h shaking at 120 rpm. After 4 h measure OD600 and adjust N. meningitidis as described in Subheading 3.3.1. As a caveat, the iron starvation significantly affects bacterial viability; therefore, the exact number of CFU/mL as a function of OD600 must be first optimized for each strain. In our hands, strain MC58 yielded 8 × 108 CFU/mL under these conditions, whereas other strains were as low as 4.5 × 107 CFU/mL. It must be considered that nonviable bacteria still contribute LOS and other agonists for pattern recognition receptors of the innate immune system [15], thereby impacting on disease pathology and, most likely, outcome of mouse infection experiments.

     
  7. 7.

    The exact correlation of CFU/mL to OD600 needs to be determined for every strain to be worked with, as significant differences can occur between strains or mutants thereof. Colony forming units should be quantified at intervals throughout the growth cycle in the culture method to be used for the infection experiments, repeated three independent times to assure reproducibility. Late phase (dense) cultures should be diluted in growth medium to calculate CFUs since the error rate is high at high optical densities.

     
  8. 8.

    When infection is done with strain MC58, 105 CFU of per mouse (i.e., in 200 μL BHI) typically cause 90% lethal infection in C57Bl/6J mice, whereas 104 CFU/mL are non-lethal. Depending on the infection strain, mouse strain used and experimental details such as N. meningitidis mutants and other variables, the optimal dose to be used in intended experiments must be determined and confirmed reproducible prior to large experiments.

     
  9. 9.

    It is important to use two distinct injection sites for the application of iron dextran solution and bacterial inoculum. Do not mix inoculum and iron dextran prior to infection, as this will substantially decrease N. meningitidis viability when mixed.

     
  10. 10.

    Alternatively, mice can also be euthanized by overdose ketamine/xylazine injection or by cervical dislocation. However, ketamine/xylazine are narcotics; therefore, additional approval for their possession and use by the according authorities may be required. Cervical dislocation interferes with sampling of cardiac blood as it leads to rupture of the carotid arteries.

     
  11. 11.

    Heparin is added to avoid clotting, which can still occur in 1:10 diluted mouse blood; do not use EDTA or citrate, as these can reduce N. meningitidis viability.

     
  12. 12.

    Bacteremia usually rises in susceptible mice that will reach the humane endpoint, and will typically decrease markedly between 3 h and 12 h in mice that will recover.

     
  13. 13.

    Usually, the levels of bacteremia or cytokines are higher in cardiac blood than from the very periphery in the tail.

     
  14. 14.

    As for the choice of N. meningitidis isolate: Thus far, most N. meningitidis strains tested colonize the CEACAM1-humanized mouse, including strains MC58, H44/76, 139M, 38VI, 860800, 90/18311, 196/87, 94/155, F1576, 8013. Strains S3131, B16B6, however, have tended to colonize less than 50% of these animals [11].

     
  15. 15.

    Iron dextran or human transferrin is not required for intranasal infection, unless factors participating in invasive disease after colonization are investigated. However, we have never observed transition of N. meningitidis from the nasopharynx to the blood, or occurrence of invasive disease after intranasal infection of normal mice. It is notable in this regard that others have observed sepsis after nasal administration of a larger volume (50 μL, which allows effective bacterial delivery into the lung) of N. meningitidis to anesthetized Balb/c mice 7–10 days after they had been nasally infected with influenza virus [16].

     
  16. 16.

    For some strains, such as 8013, BHI is actually preferred as it leads to higher colonization frequency than when prepared in PBS + 1 mM MgCl2.

     
  17. 17.

    This step is prone to create aerosols of the N. meningitidis inoculum, and droplets can be expelled from the mouse nose. Thus, it is imperative to strictly follow all rules of safe work in biosafety cabinets.

     
  18. 18.

    Optionally, this step can also be done under isoflurane anesthesia for ease of application.

     
  19. 19.

    The blood can be used for analysis of systemic effects of nasal colonization (e.g., antibody response). Generally, exsanguination is recommended to facilitate easier sample preparation in the next steps.

     
  20. 20.

    When cytokines are to be measured instead of N. meningitidis colonization, use 500 μL of PBS + 0.1% BSA and protease inhibitors added (e.g., complete protease inhibitor cocktail from Roche). Place samples immediately on ice and centrifuge at 10,000 × g at 4 °C for 5 min and then freeze the supernatant at −80 °C until analysis. Note that all downstream analyses must be done in a biosafety cabinet as presence of viable N. meningitidis in the sample cannot be ruled out unless the sample is filter sterilized.

     

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute for Hygiene and MicrobiologyUniversity of WürzburgWürzburgGermany
  2. 2.Department of Molecular GeneticsUniversity of TorontoTorontoCanada

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