An Intragastric Gavage Technique for Controlled Helicobacter Infection in Mice

Overview

This video demonstrates the establishment of Helicobacter pylori infection through the intragastric gavage technique in a mouse model. The bacterial suspension is administered via a catheter inserted through the mouth into the stomach. Inoculated bacteria colonize and establish an infection in the stomach, which can be used to study the host-pathogen interaction.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Growth and Preparation of Bacterial Inocula

1. Thaw glycerol stocks of Helicobacter pylori (H. pylori) or H. felis from -80 °C and subculture on horse blood agar (HBA) plates comprising: Blood Agar Base No.2 (see Table of Materials); a modified "Skirrow's antibiotic selective supplement" (consisting of vancomycin, 10 μg/mL; polymyxin B, 25 ng/mL; trimethoprim, 5 µg/mL; amphotericin B, 2.5 μg/mL); and 5–10% (v/v) horse blood. The bacteria grow well under microaerobic conditions in 2.5 or 3.5 L anaerobic jars containing the appropriate gas packs (see Table of Materials), at 37 °C.
   NOTE: H. pylori strains grown under these conditions must be subcultured after 1–1.5 days of incubation, whereas for H. felis, at least 2 days of incubation is generally required. Suitable culture and storage media have been described in detail previously.
2. Prepare bacterial inocula for mouse infection from early-to-mid logarithmic phase cultures. Harvest bacteria gently from agar plates by flooding each plate with 1–2 mL of brain heart infusion (BHI) broth. Aspirate suspensions from plates with Pasteur pipettes.
   1. Alternatively, prepare inocula from Helicobacter bacteria that have been propagated in BHI broth for 16–18 h. In this case, collect bacteria by low-speed centrifugation at 2,200 x g for 10 min at 4 °C.
3. Assess the viability and motility of the bacteria by examining wet mount preparations under phase contrast microscopy (100X objective). Prepare wet mounts by resuspending a loopful of bacteria in a 10–20 μL droplet of BHI broth on a glass microscope slide. In the case of H. felis, which is a much larger bacterium than H. pylori, use a hemocytometer to accurately count the number of viable bacteria. Confirm culture purity by performing a Gram stain.
   NOTE: Only use H. pylori inocula if the majority of bacteria have a bacillary or spiral shape (the morphology can vary depending on the strain). H. felis inocula should primarily contain helical-shaped bacteria. Do not use inocula if most bacteria have a coccoid morphology, as these forms are not viable and will not establish an infection in mice.
4. Estimate the number of bacteria in the inoculum by counting under phase contrast microscopy the approximate number of bacteria per field (100X objective) and by using the following guide: 1 bacterium per field = approximately 10⁶ colony-forming units (CFU) of Helicobacter/mL; 10 bacteria per field = approximately 10⁷ CFU/mL; 100 bacteria per field = approximately 10⁸ CFU/mL, etc.
   1. When using a hemocytometer, calculate CFU/mL using the following formula:
      CFU/mL = (average number of bacteria in a 4 x 4 field) x (dilution factor) x (10⁴).
5. Adjust the bacterial cell density of the inoculum to approximately 10⁷–10⁸ CFU/mL by dilution in BHI broth, if necessary.
   1. To ensure maximal bacterial viability, use inocula for intragastric gavage as soon as possible after preparation.
   2. Always confirm H. pylori cell density and viability by performing viable counting of inocula immediately after the gavage procedure (see below). This is not always possible for H. felis, as it does not usually form isolated colonies on culture media. The numbers of viable Helicobacters in inocula cannot be determined by optical density measurement (A₆₀₀) alone as this method does not discriminate between viable (i.e., bacillary/spiral/helical) and non-viable (i.e., coccoid) bacteria.
   3. Use optical density values as a means of estimating the numbers of viable H. pylori bacteria in inocula, but in this case, it is first necessary to generate a growth curve. For this, the A₆₀₀ values of H. pylori cultures are monitored over time and correlated directly against the numbers of viable bacteria, determined by plate counting.  
      NOTE: A convenient method for performing such growth curve determinations is to culture bacteria in a liquid medium (Section 1.2), using standard flat bottom tissue culture flasks placed in a 10% CO₂ incubator. The numbers of CFUs, determined from aliquots of the cultures obtained every 4–6 h over 2–3 days, are then compared to the corresponding A₆₀₀ values. Importantly, growth curves must be generated for each H. pylori strain, as these may grow at different rates and, furthermore, not all strains grow well in 10% CO₂.

2. Intragastric Gavage of Mice with Helicobacter

NOTE: This method of intragastric gavage can be applied to other bacterial species that colonize the gut e.g. S. Typhimurium, C. rodentium, Listeria monocytogenes.

1. Use 6–8-week-old, specific pathogen-free (SPF) and Helicobacter-free male or female mice. Use animals with a C57BL/6 genetic background for infection experiments with H. pylori or H. felis. In the present study, we used wild-type (WT) and genetically modified C57BL/6 mice lacking a key innate immune receptor (termed knock-out or KO animals).
   NOTE: Mice of other genetic backgrounds can also be used for Helicobacter infections, however, colonization levels and disease severity may be impacted by the type of host background.
2. Aspirate the bacterial inoculum (Step 1.5) into disposable 1 mL syringes and replace the supplied needles with 23 gauge needles onto which are affixed disposable polyethylene catheters (length, 6–8 cm; internal diameter, 0.58 mm). Fasten catheters to the needles by the application of small strips of plastic film (see Table of Materials). Alternatively, replace the needle/catheter assembly by using sterile plastic feeding tubes (20 gauge x 38 mm).
3. Physically restrain mice with a firm grip on the scruff of the neck and tail.
   NOTE: This procedure can be performed without anesthesia, or alternatively, with the use of an inhaled anesthetic, such as methoxyflurane or isoflurane.
4. Insert the catheter into the center of the open jaw and guide it in a caudal direction towards the esophagus. Extend the neck of the mouse to allow ease of access to the stomach through the esophagus (and away from the trachea) until most or all of the catheter is no longer visible and resistance is felt, corresponding to the base of the stomach. Deliver a specific aliquot, usually 100 μL per inoculation (Figure 1).
   NOTE: Mice should be gavaged with ≥ 10⁵ CFU to ensure optimal colonization and disease pathology.

Representative Results

Figure 1: Image demonstrating the oral gavage technique. A disposable 1 mL syringe and flexible catheter are used to deliver ≥10⁵ CFU of bacterial inocula to a mouse via the intragastric route. The mouse was anesthetized using methoxyflurane and held in a firm grip at the neck, allowing for access of the catheter to the stomach via the esophagus.

Materials

Name	Company	Catalog Number	Comments
Bacteriological reagents
Oxoid Blood Agar Base No.2	Thermo Fischer Scientific	CM0271B	Dissolve in deinonized water prior to sterilization
Premium Defibrinated Horse blood	Australian Ethical Biologicals	PDHB100
Bacto Brain Heart Infusion Broth	BD Bioscience	237500	Dissolve in deinonized water prior to sterilization
CampyGen gas packs	Thermo Fischer Scientific	CN0035A/CN0025A
Other reagents
Methoxyflurane (Pentrhox)	Medical Developments International	Not applicable
Equipment and plasticware
Oxoid Anaerobic Jars	Thermo Fischer Scientific	HP0011/HP0031
COPAN Pasteur Pipettes	Interpath Services	200CS01
Eppendorf 5810R centrifuge			Collect bacterial pellets by centrifugation at 2,200 rpm for 10 mins at 4°C
23g precision glide needle	BD Bioscience	301805
Parafilm M	Bemis, VWR	PM996
Portex fine bore polythene tubing	Smiths Medical	800/100/200
Plastic feeding catheters	Instech  Laboratories	FTP20-30
1 ml tuberculin luer slip disposable syringes	BD Bioscience	302100
Eppendorf micropestle for 1.2 - 2 mL tubes	Sigma Aldrich	Z317314	Autoclavable polypropylene pestles used for stomach homogenization
Sterile plastic loop	LabServ	LBSLP7202