Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.
Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing ≥105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.
Helicobacter pylori is a spiral-shaped, Gram-negative, human gastric pathogen present in all populations across the world, with infection rates in developing countries estimated to be in the order of 80%1. Although most H. pylori-infected individuals are asymptomatic, some develop more severe diseases, ranging from peptic ulceration to gastric cancer2. H. pylori-associated cancers are broadly characterized either by malignant changes in epithelial cells (GECs) or by the formation of extra-nodal lymphoid tissues in the stomach, resulting in gastric adenocarcinoma or mucosa-associated lymphoid tissue (MALT) lymphoma, respectively. H. pylori is highly adapted to survive in the harsh ecological niche of the stomach due to the presence of various virulence factors and mechanisms facilitating its adherence, growth and metabolism in this niche. In particular, virulent strains of H. pylori possess the 40 kb cag Pathogenicity Island (cagPAI) that encodes approximately 30 genes required for the production of a Type 4 secretion system (T4SS)3,4. cagPAI-positive H. pylori strains are associated with the induction of higher levels of chronic inflammation in the host, which has been implicated as an essential precursor of gastric adenocarcinoma5.
In vivo animal models, particularly mice, have been highly informative by allowing researchers to investigate the relative contributions of host, bacterial and environmental factors on H. pylori infection and disease outcome6. Studies have previously demonstrated that prolonged H. pylori infection of mice on the C57BL/6 genetic background results in the development of chronic gastritis and gland atrophy, both hallmarks of H. pylori infection7. Furthermore, infection with the related feline/canine bacterial species, H. felis, has been shown to induce MALT formation in mice with similar pathology and disease progression as seen in human MALT lymphoma8,9. The most commonly used H. pylori isolate in mouse colonization studies is the “Sydney Strain 1” (SS1) strain10, which is cagPAI+ but has a non-functional T4SS (T4SS−)11. Other widely used strains include H. pylori B128 7.13 (cagPAI+/T4SS+)12 and X47-2AL (cagPAI–/T4SS−)13. For H. felis infections, the strain CS1 (“Cat Spiral 1”, cagPAI–/T4SS−) is generally used14.
Herein, we provide a protocol describing the preparation of Helicobacter inocula for in vivo infection, the procedure for intragastric gavage of mice, as well as methods for the processing of tissues for the study of histopathological changes in the stomach. In particular, this article will focus on the histological methods used to visualize bacterial colonization and assess histopathological changes, including MALT formation, in the gastric mucosa of infected mice. Some of the methods described here may be adapted to the study of other gut pathogens such as S. Typhimurium or C. rodentium.
1. Growth and Preparation of Bacterial Inocula
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.
3. Harvesting Tissues from Mice Post-experiment
4. Confirmation of Bacterial Colonization in the Stomach Post-infection
5. Histological Analyses of Helicobacter-infected Mouse Stomach Sections
This protocol describes an oral gavage technique to achieve intragastric infection with H. pylori or H. felis in murine mouse models (Figure 1). Following euthanasia, stomachs are removed, weighed and divided into 2 equal halves comprising the antrum, body and non-glandular regions of gastric tissues (Figure 2). The non-glandular region is removed prior to performing any analyses.
Successful colonization of animals is typically confirmed by performing viable counting on H. pylori-infected gastric homogenates, and subsequently enumerating individual colonies on HBA plates (Figure 3). Alternatively, PCR is employed to verify infection with H. felis using specific, validated primers directed at a 325 bp region of the H. felis and H. pylori ureB genes (Figure 4).
Gastric tissues are processed, embedded and sectioned for downstream histological applications. The H&E staining technique is used to assess the histopathology in Helicobacter-infected mice. In the current example, WT C57BL/6 mice display moderate signs of inflammation, including hyperplasia (enlarged mucosa) and gland atrophy at 6 months’ post-infection with H. felis. The presence of cellular infiltrates can also be observed in the sub-mucosa. Interestingly, however, more severe inflammation is observed in KO mice at the same time point, with the additional presence of lymphoid follicles located in close proximity to cellular infiltrates (Figure 5). Finally, H. felis bacteria are observed in Giemsa-stained sections of infected mouse stomachs (Figure 6).
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.
Figure 2: Harvesting of mouse spleens and stomachs post-infection. Mouse stomachs were harvested post-euthanasia and their contents removed by scraping with a scalpel and washing in sterile PBS. The tissues were then weighed and flattened on a cotton sheet to reveal 2 equal halves, each comprising the gastric antrum, body and non-glandular regions; Scale bar = 10 mm.
Figure 3: Viable counts on H. pylori-infected mouse stomachs. Mice on the C57BL/6 background were inoculated with 107 CFU of H. pylori SS1 and left for 8 weeks. (A) Dilutions of each gastric homogenate are plated onto a half (or third) of an HBA plate and bacterial loads assessed by enumerating 10-100 individual colonies. The left half of the plate shows a pure culture of H. pylori bacteria. (B) The presence of contaminating bacteria from the mouse gastric microbiota (left) or large numbers of H. pylori colonies (right) can complicate the enumeration of H. pylori CFUs. (C) Common examples of contaminating bacteria in gastric homogenate samples. Scale bar = 1.7 cm. Please click here to view a larger version of this figure.
Figure 4: PCR detection of H. felis infection in gastric biopsies using oligonucleotides targeting the ureB gene. A specific oligonucleotide pair was designed to recognize and bind to homologous sequences in both H. felis and H. pylori ureB genes. These primers were validated using genomic DNA from H. pylori SS1 (lane 2) or H. felis (lane 3). Deionized water was included as a negative control (lane 1).
Figure 5: Representative images of H&E-stained stomach sections from WT and KO mice at 6 months’ post-infection with H. felis. Paraffin-embedded tissue sections were stained with H&E. WT mice receiving BHI broth alone (control) had a normal gastric epithelium and no significant inflammation. In contrast, WT animals with chronic H. felis infection displayed moderate levels of inflammation and mucosal thickening which was further exacerbated in H. felis-infected KO animals. Tissue sections from H. felis-infected KO mice exhibited the presence of mucosal lymphoid follicles (*), cellular infiltrates (→), gland atrophy (▶) and hyperplasia. Scale bar = 100 μm. Please click here to view a larger version of this figure.
Figure 6: Representative images of Giemsa-stained gastric sections from C57BL/6 WT mice at 3 months’ post-infection with H. felis. Paraffin-embedded tissue sections were stained with Giemsa. Arrows indicate the presence of H. felis in the gastric glands. Scale bar = 200 μm.
This protocol describes the use of an in vivo mouse model for Helicobacter infection. The critical steps of the procedure are the: 1) preparation of Helicobacter inocula containing viable and motile bacteria; 2) delivery of the appropriate numbers of bacteria to the mouse via intragastric gavage; 3) detection of bacterial infection by colony counting and/or PCR; and 4) processing of gastric tissues to enable the assessment of histopathology in infected stomachs. Further suggestions for modifications, troubleshooting and technical considerations are discussed below.
The method of growing Helicobacter spp. using Blood Agar Base no. 2 supplemented with horse blood has been well established in our laboratory. However, alternate agar bases such as Brucella agar and Columbia blood agar can also be used22. It is important to ensure that only sterile glassware that is free of detergent is used to prepare the growth medium. Furthermore, to obtain optimal growth, H. pylori bacteria should be routinely subcultured on agar plates that have residual moisture and are not dry. When preparing Helicobacter spp. inocula for infection, it is vital to subculture H. pylori and H. felis strains every 1–1.5 or 2 days, respectively, to ensure bacterial viability. At every subculture, bacteria should be assessed for their viability and motility by phase contrast microscopy. A urease assay can also be routinely employed to discriminate between gastric Helicobacter spp. and other bacteria23, however, it is important to realize that this assay detects both viable and non-viable Helicobacter bacteria. Following inoculation of animals, viable counts on Helicobacter suspensions must be performed to quantitate numbers of viable bacteria used for infection. As H. felis does not reproducibly form isolated colonies on agar medium15, estimation of bacterial numbers is performed using phase contrast microscopy. Quantification of bacterial numbers by optical density measurement (A600) alone is inaccurate as this method does not discriminate between viable and non-viable bacteria. This method should not be used in Helicobacter research without rigorous optimization, as described above (Section 1.5).
When performing Helicobacter infection studies, it is crucial to consider the optimal mouse and Helicobacter strain, as well as the length of infection, to suit the purpose of the experiment. It is also essential to regularly confirm that the animals used for experimentation are indeed Helicobacter-free using genus-specific PCR primers24. The presence of other enteric Helicobacter species, such as Helicobacter bilis, Helicobacter hepaticus or Helicobacter muridarum, may alter the disease susceptibility of mice and introduce confounding factors into in vivo studies25,26. It is also advisable to include a mock treatment control group of animals (i.e., fed broth only) in initial screening experiments to investigate the effects of the normal microbiota on Helicobacter colonization and pathogenesis.
Post-euthanasia, H. pylori colonization in murine stomachs can be measured by viable counting. HBA plates used for colony counts should be supplemented with bacitracin and naladixic acid in addition to the modified Skirrow’s selective supplement, to restrict the growth of bacterial species from the normal gastric microbiota and hence prevent contamination27. H. felis does not always form colonies, but instead tends to form swarming growth on agar plates15,28. Therefore, PCR and qPCR are normally employed to determine the presence and levels of H. felis, respectively29,30. In section 4.2, we introduced a simple and quick PCR method to confirm colonization by H. felis in the murine stomach using a pair of primers, which have been validated to target a 325 bp region of H. felis and H. pylori ureB genes. Using the PCR conditions described above, it is possible to discriminate between infection by these gastric Helicobacter spp. and urease-producing enterohepatic Helicobacters. Other genes that have been validated for PCR detection of gastric Helicobacter infection include the 16s rRNA and flagellin B (flaB) genes15,29,30.
Finally, we have described the use of two powerful staining techniques: H&E staining, to assess histopathological changes in the stomach post-infection; and Giemsa staining, to detect H. felis infection. To obtain optimal staining, it is essential to ensure that tissues have been preserved, processed and embedded in the correct orientation. Additionally, only freshly prepared solutions and filtered stains must be used during this process. Tissue sections can be stored indefinitely and utilized for more specific analysis of gastric pathology via immunofluorescence or immunohistochemistry. Some other common measures of gastric inflammation and disease include: immune cell recruitment (anti-CD45 staining); mucosal thickening/destruction (Periodic Acid Schiff/Alcian blue staining); epithelial cell proliferation (proliferating cell nuclear antigen, PCNA/Bromodeoxyuridine, BrDU staining); or cellular apoptosis (TUNEL staining). The lymphoid follicles observed in the H&E-stained tissues of H. felis-infected mice can be confirmed by immunohistochemistry, using antibodies directed against B (B220+) and T cell (CD3+) antigens31.
In summary, animal models of bacterial disease provide valuable tools in the field of infection biology. The protocols of intragastric gavage and processing of stomach tissues provided here may be adapted to mouse infection models involving other enteric pathogens.
The authors have nothing to disclose.
The authors would like to thank Ms. A. De Paoli and Ms. Georgie Wray-McCann for technical assistance. The authors acknowledge use of the facilities and technical assistance of Monash Histology Platform, Department of Anatomy and Developmental Biology, Monash University. The laboratory is supported by funding from the National Health and Medical Research Council (NHMRC) to RLF (APP1079930, APP1107930). RLF is supported by a Senior Research Fellowship from the NHMRC (APP1079904). KD and MC are both supported by Monash Graduate Scholarships. KD is also supported by the Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, while MC has an International Postgraduate Scholarship from the Faculty of Medicine, Nursing and Health Sciences, Monash University. Research at the Hudson Institute of Medical Research is supported by the Victorian Government’s Operational Infrastructure Support Program.
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 | |
Histological reagents | |||
Formalin, neutral buffered, 10% | Sigma Aldrich | HT501128 | |
Absolute alcohol, 100% Denatured | ChemSupply | AL048-20L-P | |
Isopropanol (2-propanol) | Merck | 100995 | |
Xylene (sulphur free) | ChemSupply | XT003-20L | |
Mayer's Haematoxylin | Amber Scientific | MH-1L | Filter before use |
Eosin, Aqueous Stain | Amber Scientific | EOCA-1L | Filter before use |
Wright-Giemsa Stain, modified | Sigma Aldrich | WG80-2.5L | Dilute before use (20% Giemsa, 80% deionized water) |
Histolene | Grale Scientific | 11031/5 | |
DPX mounting medium | VWR | 1.00579.0500 | |
Molecular biology reagents | |||
Qubit dsDNA BR Assay Kit | Thermo Fischer Scientific | Q32850 | |
Oligonucleotides | Sigma Aldrich | The annealing temperature of ureB primers used in this study is 61°C | |
GoTaq Flexi DNA Polymerase | Promega | M8291 | Kit includes 10X PCR buffer and Magnesium Chloride |
dNTPs | Bioline | BIO-39028 | Dilute to 10mM in sterile nuclease free water before use |
Molecular Grade Agarose | Bioline | BIO-41025 | |
Sodium Hydrogen Carbonate | Univar (Ajax Fine Chemicals) | A475-500G | |
Magnesium Sulphate Heptahydrate | Chem-Supply | MA048-500G | |
Antibiotics | |||
Vancomycin | Sigma Aldrich | V2002-1G | Dissolve in deionized water |
Polymyxin B | Sigma Aldrich | P4932-5MU | Dissolve in deionized water |
Trimethoprim (≥98% HPLC) | Sigma Aldrich | T7883 | Dissolve in 100% (absolute) Ethanol |
Amphotericin | Amresco (Astral Scientific) | E437-100MG | Dissolve in deionized water |
Bacitracin from Bacillus licheniformis | Sigma Aldrich | B0125 | Dissolve in deionized water |
Naladixic acid | Sigma Aldrich | N8878 | Dissolve in deionized water |
Other reagents | |||
Methoxyflurane (Pentrhox) | Medical Developments International | Not applicable | |
Paraffin Wax | Paraplast Plus, Leica Biosystems | 39601006 | |
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 |
GentleMACs Dissociator | Miltenyi Biotec | 130-093-235 | Use a pre-set gentleMACS Programs for mouse stomach tissue |
M Tubes (orange cap) | Miltenyi Biotec | 30-093-236 | |
Qubit Fluorometer | Thermo Fischer Scientific | Q33216 | |
Sterile plastic loop | LabServ | LBSLP7202 | |
Cold Plate, Leica EG1160 Embedding System | Leica Biosystems | Not applicable | |
Tissue-Tek Base Mould System, Base Mold 38 x 25 x 6 | Sakura, Alphen aan den Rijn | 4124 | |
Tissue-Tek III Uni-Casette System | Sakura, Alphen aan den Rijn | 4170 | |
Microtome, Leica RM2235 | Leica Biosystems | ||
Charged SuperFrost Plus glass slides | Menzel Glaser, Thermo Fischer Scientific | 4951PLUS4 |