Name: mouse models of helicobacter infection and gastric pathologies
Uploaded: 2018-10-18T13:00:00
Description: Watch this Scientific Journal Video about Mouse Models Of Helicobacter Infection And Gastric Pathologies at JoVE.com

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&#8217;s Operational Infrastructure Support Program.

1. Growth and Preparation of Bacterial Inocula
<ol>
	<li>Thaw glycerol stocks of H. pylori or H. felis15 from -80 &#176;C and subculture on horse blood agar (HBA) plates comprising: Blood Agar Base No.2 (see Table of Materials); a modified &#8220;Skirrow&#8217;s antibiotic selective supplement&#8221; (consisting of vancomycin, 10 &#956;g/mL; polymyxin B, 25 ng/mL; trimethoprim, 5 &#181;g/mL; amphotericin B, 2.5 &#956;g/mL); and 5&#8211;10% (v/v) horse blood<sup...

<ol>
	<li>Goh, K. L., Chan, W. K., Shiota, S., Yamaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Helicobacter+pylori+infection+and+public+health+implications.">Epidemiology of Helicobacter pylori infection and public health implications.</a> Helicobacter. 16, 1-9 (2011).</li><li>Montecucco, C., Rappuoli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+dangerously:+how+Helicobacter+pylori+survives+in+the+human+stomach.">Living dangerously: how Helicobacter pylori survives in the human stomach.</a> Nature Reviews Molecular Cell Biology. 2 (6), 457-466 (2001).</li><li>Akopyants, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+the+cag+pathogenicity+island+of+Helicobacter+pylori.">Analyses of the cag pathogenicity island of Helicobacter pylori.</a> Molecular Microbiology. 28 (1), 37-53 (1998).</li><li>Censini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cag,+a+pathogenicity+island+of+Helicobacter+pylori,+encodes+type+I-specific+and+disease-associated+virulence+factors.">cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (25), 14648-14653 (1996).</li><li>Peek, R. M., Fiske, C., Wilson, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+innate+immunity+in+Helicobacter+pylori-induced+gastric+malignancy.">Role of innate immunity in Helicobacter pylori-induced gastric malignancy.</a> Physiological Reviews. 90 (3), 831-858 (2010).</li><li>O'Rourke, J. L., Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+Helicobacter+pylori+infection+and+disease.">Animal models of Helicobacter pylori infection and disease.</a> Microbes and Infection. 5 (8), 741-748 (2003).</li><li>Sakagami, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrophic+gastric+changes+in+both+Helicobacter+felis+and+Helicobacter+pylori+infected+mice+are+host+dependent+and+separate+from+antral+gastritis.">Atrophic gastric changes in both Helicobacter felis and Helicobacter pylori infected mice are host dependent and separate from antral gastritis.</a> Gut. 39 (5), 639-648 (1996).</li><li>Correa, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+and+gastric+carcinogenesis.">Helicobacter pylori and gastric carcinogenesis.</a> The American Journal of Surgical Pathology. 19, S37-S43 (1995).</li><li>Enno, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALToma-like+lesions+in+the+murine+gastric+mucosa+after+long-term+infection+with+Helicobacter+felis.+A+mouse+model+of+Helicobacter+pylori-induced+gastric+lymphoma.">MALToma-like lesions in the murine gastric mucosa after long-term infection with Helicobacter felis. A mouse model of Helicobacter pylori-induced gastric lymphoma.</a> The American Journal of Pathology. 147 (1), 217-222 (1995).</li><li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+mouse+model+of+Helicobacter+pylori+infection:+introducing+the+Sydney+strain.">A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain.</a> Gastroenterology. 112 (4), 1386-1397 (1997).</li><li>Crabtree, J. E., Ferrero, R. L., Kusters, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+colonizing+Helicobacter+pylori+strain+SS1+may+lack+a+functional+cag+pathogenicity+island.">The mouse colonizing Helicobacter pylori strain SS1 may lack a functional cag pathogenicity island.</a> Helicobacter. 7 (2), 139-140 (2002).</li><li>Israel, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+strain-specific+differences+in+genetic+content,+identified+by+microarray,+influence+host+inflammatory+responses.">Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses.</a> Journal of Clinical Investigation. 107 (5), 611-620 (2001).</li><li>Fox, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori-induced+gastritis+in+the+domestic+cat.">Helicobacter pylori-induced gastritis in the domestic cat.</a> Infection and Immunity. 63 (7), 2674-2681 (1995).</li><li>Lee, A., Hazell, S. L., O'Rourke, J., Kouprach, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+spiral-shaped+bacterium+from+the+cat+stomach.">Isolation of a spiral-shaped bacterium from the cat stomach.</a> Infection and Immunity. 56 (11), 2843-2850 (1988).</li><li>Ferrero, R. L., Wilson, J. E., Sutton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+Helicobacter-induced+gastric+cancer:+use+of+cocarcinogens.">Mouse models of Helicobacter-induced gastric cancer: use of cocarcinogens.</a> Methods in Molecular Biology. 921, 157-173 (2012).</li><li>Ferrero, R. L., Thiberge, J. M., Huerre, M., Labigne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+of+specific-pathogen-free+mice+to+chronic+Helicobacter+pylori+(strain+SS1)+infection.">Immune responses of specific-pathogen-free mice to chronic Helicobacter pylori (strain SS1) infection.</a> Infection and Immunity. 66 (4), 1349-1355 (1998).</li><li>Blanchard, T. G., Nedrud, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+of+Helicobacter+species.">Laboratory maintenance of Helicobacter species.</a> Current Protocols in Microbiology. , (2012).</li><li>Kim, J. S., Chang, J. H., Chung, S. I., Yum, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+host+genetic+background+on+immune+responses+to+Helicobacter+pylori+infection+and+therapeutic+vaccine+efficacy.">Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy.</a> FEMS Immunology and Medical Microbiology. 31 (1), 41-46 (2001).</li><li>Nedrud, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+genetic+influence+on+the+innate+inflammatory+response+to+helicobacter+infection+of+the+gastric+mucosa.">Lack of genetic influence on the innate inflammatory response to helicobacter infection of the gastric mucosa.</a> Frontiers in Immunology. 3, 181 (2012).</li><li>Cai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+felis+eradication+restores+normal+architecture+and+inhibits+gastric+cancer+progression+in+C57BL/6+mice.">Helicobacter felis eradication restores normal architecture and inhibits gastric cancer progression in C57BL/6 mice.</a> Gastroenterology. 128 (7), 1937-1952 (2005).</li><li>Ferrero, R. L., Labigne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning,+expression+and+sequencing+of+Helicobacter+felis+urease+genes.">Cloning, expression and sequencing of Helicobacter felis urease genes.</a> Molecular Microbiology. 9 (2), 323-333 (1993).</li><li>Stevenson, T. H., Castillo, A., Lucia, L. M., Acuff, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Helicobacter+pylori+in+various+liquid+and+plating+media.">Growth of Helicobacter pylori in various liquid and plating media.</a> Letters in Applied Microbiology. 30 (3), 192-196 (2000).</li><li>Uotani, T., Graham, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+Helicobacter+pylori+using+the+rapid+urease+test.">Diagnosis of Helicobacter pylori using the rapid urease test.</a> Annals of Translational Medicine. 3 (1), 9 (2015).</li><li>Riley, L. K., Franklin, C. L., Hook, R. R., Besch-Williford, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+murine+helicobacters+by+PCR+and+restriction+enzyme+analyses.">Identification of murine helicobacters by PCR and restriction enzyme analyses.</a> Journal of Clinical Microbiology. 34 (4), 942-946 (1996).</li><li>Chaouche-Drider, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+commensal+Helicobacter+sp.+of+the+rodent+intestinal+flora+activates+TLR2+and+NOD1+responses+in+epithelial+cells.">A commensal Helicobacter sp. of the rodent intestinal flora activates TLR2 and NOD1 responses in epithelial cells.</a> PLoS One. 4 (4), e5396 (2009).</li><li>Fox, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+bilis:+bacterial+provocateur+orchestrates+host+immune+responses+to+commensal+flora+in+a+model+of+inflammatory+bowel+disease.">Helicobacter bilis: bacterial provocateur orchestrates host immune responses to commensal flora in a model of inflammatory bowel disease.</a> Gut. 56 (7), 898-900 (2007).</li><li>McGee, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+enhances+Helicobacter+pylori+resistance+to+antibiotics+and+LL-37.">Cholesterol enhances Helicobacter pylori resistance to antibiotics and LL-37.</a> Antimicrobial Agents and Chemotherapy. 55 (6), 2897-2904 (2011).</li><li>Viala, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nod1+responds+to+peptidoglycan+delivered+by+the+Helicobacter+pylori+cag+pathogenicity+island.">Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island.</a> Nature Immunology. 5 (11), 1166-1174 (2004).</li><li>Kong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+specific+PCR+method+to+detect+Helicobacter+felis+in+a+conventional+mouse+model.">A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model.</a> Clinical and Vaccine Immunology. 3 (1), 73-78 (1996).</li><li>Ng, G., Every, A., McGuckin, M., Sutton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Helicobacter+felis+colonization+in+male+129/Sv+mice+fails+to+suppress+gastritis.">Increased Helicobacter felis colonization in male 129/Sv mice fails to suppress gastritis.</a> Gut Microbes. 2 (6), 358-360 (2011).</li><li>Ferrero, R. L., Ave, P., Radcliff, F. J., Labigne, A., Huerre, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outbred+mice+with+long-term+Helicobacter+felis+infection+develop+both+gastric+lymphoid+tissue+and+glandular+hyperplastic+lesions.">Outbred mice with long-term Helicobacter felis infection develop both gastric lymphoid tissue and glandular hyperplastic lesions.</a> The Journal of Pathology. 191 (3), 333-340 (2000).</li></ol>

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 modifica...

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 &#8220;Sydney Strain 1&#8221; (SS1) strain10, which is cagPAI+ but has a non-functional T4SS (T4SS&#8722;)11. Other widely used strains include H. pylori B128 7.13 (cagPAI+/T4SS+)12 and X47-2AL (cagPAI-/T4SS&#8722;)13. For H. felis infections, the strain CS1 (&#8220;Cat Spiral 1&#8221;, cagPAI-/T4SS&#8722;) 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.

<table>
	<tbody>
		<tr>
			<td>Bacteriological reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxoid Blood Agar Base No.2</td>
			<td>Thermo Fischer Scientific</td>
			<td>CM0271B</td>
			<td>Dissolve in deinonized water prior to sterilization</td>
		</tr>
		<tr>
			<td>Premium Defibrinated Horse blood</td>
			<td>Australian Ethical Biologicals</td>
			<td>PDHB100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion Broth</td>
			<td>BD Bioscience</td>
			<td>237500</td>
			<td>Dissolve in deinonized water prior to sterilization</td>
		</tr>
		<tr>
			<td>CampyGen gas packs</td>
			<td>Thermo Fischer Scientific</td>
			<td>CN0035A/CN0025A</td>
			<td></td>
		</tr>
		<tr>
			<td>Histological reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin, neutral buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute alcohol, 100% Denatured</td>
			<td>ChemSupply</td>
			<td>AL048-20L-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (2-propanol)</td>
			<td>Merck</td>
			<td>100995</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene (sulphur free)</td>
			<td>ChemSupply</td>
			<td>XT003-20L</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#39;s Haematoxylin</td>
			<td>Amber Scientific</td>
			<td>&#160;MH-1L</td>
			<td>Filter before use</td>
		</tr>
		<tr>
			<td>Eosin, Aqueous Stain</td>
			<td>Amber Scientific</td>
			<td>EOCA-1L</td>
			<td>Filter before use</td>
		</tr>
		<tr>
			<td>Wright-Giemsa Stain, modified</td>
			<td>Sigma Aldrich</td>
			<td>WG80-2.5L</td>
			<td>Dilute before use (20% Giemsa, 80% deionized water)</td>
		</tr>
		<tr>
			<td>Histolene</td>
			<td>Grale Scientific</td>
			<td>11031/5</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mounting medium</td>
			<td>VWR</td>
			<td>1.00579.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>Q32850</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligonucleotides</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>The annealing temperature of ureB primers used in this study is 61&#176;C</td>
		</tr>
		<tr>
			<td>GoTaq Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>&#160;M8291</td>
			<td>Kit includes 10X PCR buffer and Magnesium Chloride</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Bioline</td>
			<td>BIO-39028</td>
			<td>Dilute to 10mM in sterile nuclease free water before use</td>
		</tr>
		<tr>
			<td>Molecular Grade Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydrogen Carbonate</td>
			<td>Univar (Ajax Fine Chemicals)</td>
			<td>A475-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulphate Heptahydrate</td>
			<td>Chem-Supply</td>
			<td>MA048-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vancomycin</td>
			<td>Sigma Aldrich</td>
			<td>V2002-1G</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Polymyxin B</td>
			<td>Sigma Aldrich</td>
			<td>P4932-5MU</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Trimethoprim (&#8805;98% HPLC)</td>
			<td>Sigma Aldrich</td>
			<td>T7883</td>
			<td>Dissolve in 100% (absolute) Ethanol</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Amresco (Astral Scientific)</td>
			<td>E437-100MG</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Bacitracin from Bacillus licheniformis</td>
			<td>Sigma Aldrich</td>
			<td>B0125</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Naladixic acid</td>
			<td>Sigma Aldrich</td>
			<td>N8878</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Other reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxyflurane (Pentrhox)</td>
			<td>Medical Developments International</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Wax</td>
			<td>Paraplast Plus, Leica Biosystems</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment and plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxoid Anaerobic Jars</td>
			<td>Thermo Fischer Scientific</td>
			<td>HP0011/HP0031</td>
			<td></td>
		</tr>
		<tr>
			<td>COPAN Pasteur Pipettes</td>
			<td>Interpath Services</td>
			<td>200CS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5810R centrifuge</td>
			<td></td>
			<td></td>
			<td>Collect bacterial pellets by centrifugation at 2,200 rpm for 10 mins at 4&#176;C</td>
		</tr>
		<tr>
			<td>23g precision glide needle</td>
			<td>BD Bioscience</td>
			<td>301805</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis, VWR</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Portex fine bore polythene tubing</td>
			<td>Smiths Medical</td>
			<td>800/100/200</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic feeding catheters</td>
			<td>Instech&#160; Laboratories</td>
			<td>FTP20-30</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml tuberculin luer slip disposable syringes</td>
			<td>BD Bioscience</td>
			<td>302100</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf micropestle for 1.2 - 2 mL tubes</td>
			<td>Sigma Aldrich</td>
			<td>Z317314</td>
			<td>Autoclavable polypropylene pestles used for stomach homogenization</td>
		</tr>
		<tr>
			<td>GentleMACs Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Use a pre-set gentleMACS Programs for mouse stomach tissue</td>
		</tr>
		<tr>
			<td>M Tubes (orange cap)</td>
			<td>Miltenyi Biotec</td>
			<td>30-093-236</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Qubit Fluorometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>Q33216</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic loop</td>
			<td>LabServ</td>
			<td>LBSLP7202</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold Plate, Leica EG1160 Embedding System</td>
			<td>Leica Biosystems</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Base Mould System, Base Mold 38 x 25 x 6</td>
			<td>Sakura, Alphen aan den Rijn</td>
			<td>4124</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek III Uni-Casette System</td>
			<td>Sakura, Alphen aan den Rijn</td>
			<td>4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome, Leica RM2235</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charged SuperFrost Plus glass slides</td>
			<td>Menzel Glaser, Thermo Fischer Scientific</td>
			<td>4951PLUS4</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse models of helicobacter infection and gastric pathologies

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 &#8805;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&#38;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.

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 i...

Watch this Scientific Journal Video about Mouse Models Of Helicobacter Infection And Gastric Pathologies at JoVE.com

Mouse Models Of Helicobacter Infection And Gastric Pathologies

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.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

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 ...

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Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a common skin disease that manifests as a local rash, redness, and skin lesions. The occurrence and development of ACD are influenced by both genetic and environmental factors. Although many scholars have constructed a series of models of ACD in recent years, the experimental protocols of these models are all different, which makes it difficult for readers to establish them well. Therefore, a stable and efficient animal model is of great significance to further study the pathogenesis of atopic dermatitis. In this study, we detail a modeling method using 1-fluoro-2,4-dinitrobenzene (DNFB) to induce ACD-like symptoms in the ears of mice and describe several methods for assessing the severity of dermatitis during modeling. This experimental protocol has been successfully applied in some experiments and has a certain promotional role in the field of ACD research.

Here, we describe the methods for inducing allergic contact dermatitis in mouse ears by 1-fluoro-2,4-dinitrobenzene (DNFB) and how to evaluate the severity of allergic contact dermatitis.

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...