The authors would like to thank Trevor Lawley at the Wellcome Trust Sanger Institute for C. difficile R20291 spores and James S. Guy at the North Carolina State University College of Veterinary Medicine for Vero cells, both utilized in this manuscript. Animal histopathology was performed in the LCCC Animal Histopathology Core Facility at the University of North Carolina at Chapel Hill, with special assistance from Traci Raley and Amanda Brown. The LCCC Animal Histopathology Core is supported in part by an NCI Center Core Support Grant (2P30CA016086-40) to the UNC Lineberger Comprehensive Cancer Center. We would also like to thank Vincent Young, Anna Seekatz, Jhansi Leslie, and Cassie Schumacher for helpful discussions on the Vero cell cytotoxicity assay protocol. JAW is funded by the Ruth L. Kirschstein National Research Service Award Research Training grant T32OD011130 by NIH. CMT is funded by the career development award in metabolomics grant K01GM109236 by the NIGMS of the NIH.

<ol>
	<li>Lessa, F. C., 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=Burden+of+Clostridium+difficile+Infection+in+the+United+States.">Burden of Clostridium difficile Infection in the United States.</a> New England Journal of Medicine. 372, 825-834 (2015).</li><li>Gerding, D. N., Lessa, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+Clostridium+difficile+infection+inside+and+outside+health+care+institutions.">The epidemiology of Clostridium difficile infection inside and outside health care institutions.</a> Infect Dis Clin North Am. 29, 37-50 (2015).</li><li>Dubberke, E. R., Olsen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+Clostridium+difficile+on+the+healthcare+system.">Burden of Clostridium difficile on the healthcare system.</a> Clin Infect Dis. 55, 88-92 (2012).</li><li>Kociolek, L. K., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakthroughs+in+the+treatment+and+prevention+of+Clostridium+difficile+infection.">Breakthroughs in the treatment and prevention of Clostridium difficile infection.</a> Nat Rev Gastroenterol Hepatol. , (2016).</li><li>Kelly, C. P., LaMont, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile--more+difficult+than+ever.">Clostridium difficile--more difficult than ever.</a> N Engl J Med. 359, 1932-1940 (2008).</li><li>Louie, T. 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=Fidaxomicin+versus+vancomycin+for+Clostridium+difficile+infection.">Fidaxomicin versus vancomycin for Clostridium difficile infection.</a> N Engl J Med. 364, 422-431 (2011).</li><li>Bartlett, J. G., Onderdonk, A. B., Cisneros, R. L., Kasper, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clindamycin-associated+colitis+due+to+a+toxin-producing+species+of+Clostridium+in+hamsters.">Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters.</a> The Journal of infectious diseases. 136, 701-705 (1977).</li><li>Kelly, M. 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=Improving+the+reproducibility+of+the+NAP1/B1/027+epidemic+strain+R20291+in+the+hamster+model+of+infection.">Improving the reproducibility of the NAP1/B1/027 epidemic strain R20291 in the hamster model of infection.</a> Anaerobe. , (2016).</li><li>Kuehne, S. 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=Importance+of+toxin+A,+toxin+B,+and+CDT+in+virulence+of+an+epidemic+Clostridium+difficile+strain.">Importance of toxin A, toxin B, and CDT in virulence of an epidemic Clostridium difficile strain.</a> The Journal of infectious diseases. 209, 83-86 (2014).</li><li>Bartlett, J. G., Onderdonk, A. B., Cisneros, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clindamycin-associated+colitis+in+hamsters:+protection+with+vancomycin.">Clindamycin-associated colitis in hamsters: protection with vancomycin.</a> Gastroenterology. 73, 772-776 (1977).</li><li>Chen, 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=A+mouse+model+of+Clostridium+difficile-associated+disease.">A mouse model of Clostridium difficile-associated disease.</a> Gastroenterology. 135, 1984-1992 (2008).</li><li>Lawley, T. D., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+models+to+study+Clostridium+difficile+infection+and+transmission.">Murine models to study Clostridium difficile infection and transmission.</a> Anaerobe. 24, 94-97 (2013).</li><li>Best, E. L., Freeman, J., Wilcox, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+the+study+of+Clostridium+difficile+infection.">Models for the study of Clostridium difficile infection.</a> Gut Microbes. 3, 145-167 (2012).</li><li>Reeves, A. E., 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=The+interplay+between+microbiome+dynamics+and+pathogen+dynamics+in+a+murine+model+of+Clostridium+difficile+Infection.">The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile Infection.</a> Gut Microbes. 2, 145-158 (2014).</li><li>Owens, R. C., Donskey, C. J., Gaynes, R. P., Loo, V. G., Muto, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial-associated+risk+factors+for+Clostridium+difficile+infection.">Antimicrobial-associated risk factors for Clostridium difficile infection.</a> Clin Infect Dis. 46, 19-31 (2008).</li><li>Theriot, C. M., 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=Cefoperazone-treated+mice+as+an+experimental+platform+to+assess+differential+virulence+of+Clostridium+difficile+strains.">Cefoperazone-treated mice as an experimental platform to assess differential virulence of Clostridium difficile strains.</a> Gut Microbes. 2, 326-334 (2011).</li><li>Martin, J. S., Monaghan, T. M., Wilcox, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+epidemiology,+diagnosis+and+understanding+transmission.">Clostridium difficile infection: epidemiology, diagnosis and understanding transmission.</a> Nat Rev Gastroenterol Hepatol. , (2016).</li><li>Connor, J. R., Johnson, S., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection+caused+by+the+epidemic+BI/NAP1/027+strain.">Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain.</a> Gastroenterology. 136, 1913-1924 (2009).</li><li>He, M., 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=Emergence+and+global+spread+of+epidemic+healthcare-associated+Clostridium+difficile.">Emergence and global spread of epidemic healthcare-associated Clostridium difficile.</a> Nat Genet. 45, 109-113 (2013).</li><li>Perez, J., Springthorpe, V. S., Sattar, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clospore:+a+liquid+medium+for+producing+high+titers+of+semi-purified+spores+of+Clostridium+difficile.">Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile.</a> Journal of AOAC International. 94, 618-626 (2011).</li><li>Sorg, J. A., Dineen, S. S. <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+Clostridium+difficile.">Laboratory maintenance of Clostridium difficile.</a> Curr Protoc Microbiol. , (2009).</li><li>Edwards, A. N., Suarez, J. M., McBride, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+and+maintaining+Clostridium+difficile+in+an+anaerobic+environment.">Culturing and maintaining Clostridium difficile in an anaerobic environment.</a> Journal of visualized experiments : JoVE. , e50787 (2013).</li><li>George, W. L., Sutter, V. L., Citron, D., Finegold, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+and+differential+medium+for+isolation+of+Clostridium+difficile.">Selective and differential medium for isolation of Clostridium difficile.</a> J Clin Microbiol. 9, 214-219 (1979).</li><li>Knoblaugh, S., Randolph-Habecker, J., Rath, S., Dintzis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparative Anatomy and Histology. , 15-40 (2012).</li><li>Ammerman, N. C., Beier-Sexton, M., Azad, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+maintenance+of+Vero+cell+lines.">Growth and maintenance of Vero cell lines.</a> Curr Protoc Microbiol. , (2008).</li><li>Theriot, C. M., 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=Antibiotic-induced+shifts+in+the+mouse+gut+microbiome+and+metabolome+increase+susceptibility+to+Clostridium+difficile+infection.">Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection.</a> Nat Commun. 5, 3114 (2014).</li><li>Koenigsknecht, M. 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=Dynamics+and+establishment+of+Clostridium+difficile+infection+in+the+murine+gastrointestinal+tract.">Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract.</a> Infect Immun. 83, 934-941 (2015).</li><li>Theriot, C., Bowman, A., Young, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic-Induced+Alterations+of+the+Gut+Microbiota+Alter+Secondary+Bile+Acid+Production+and+Allow+for+Clostridium+difficile+Spore+Germination+and+Outgrowth+in+the+Large+Intestine.">Antibiotic-Induced Alterations of the Gut Microbiota Alter Secondary Bile Acid Production and Allow for Clostridium difficile Spore Germination and Outgrowth in the Large Intestine.</a> mSphere. 1, 00045 (2016).</li><li>Xiao, 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+catalog+of+the+mouse+gut+metagenome.">A catalog of the mouse gut metagenome.</a> Nature biotechnology. 33, 1103-1108 (2015).</li><li>Leslie, J. 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=Persistence+and+toxin+production+by+Clostridium+difficile+within+human+intestinal+organoids+result+in+disruption+of+epithelial+paracellular+barrier+function.">Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function.</a> Infect Immun. 83, 138-145 (2015).</li><li>Stabler, R. 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=Comparative+genome+and+phenotypic+analysis+of+Clostridium+difficile+027+strains+provides+insight+into+the+evolution+of+a+hypervirulent+bacterium.">Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium.</a> Genome Biol. 10, 102 (2009).</li><li>Valiente, E., Dawson, L. F., Cairns, M. D., Stabler, R. A., Wren, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+new+PCR+ribotypes+from+the+hypervirulent+Clostridium+difficile+027+lineage.">Emergence of new PCR ribotypes from the hypervirulent Clostridium difficile 027 lineage.</a> J Med Microbiol. 61, 49-56 (2012).</li></ol>

The authors have nothing to disclose at this time.

This protocol characterizes the clinical course, including weight loss, bacterial colonization, toxin cytotoxicity, and histopathological changes in the gastrointestinal tract, of antibiotic-treated mice challenged with C. difficile R20291 spores. There are several critical steps within the protocol where attention to detail is essential. Accurate calculation of the C. difficile spore inoculum is critical. This calculation is based on the original C. difficile spore stock enumeration, which should be consistent over multiple spore enumerations prior to starting this protocol. If the inoculum calculation is erroneous, it will result in an inaccurate inoculation dose of the mice. On the same note, preparation and dilutions of C. difficile spores based on these calculations should be prepared with care. Any dilution errors will result in an inaccurate starting spore inoculum. Lastly, precautions should be taken to avoid contamination of antibiotic-treated control mice (not inoculated with C. difficile spores). Cefoperazone-treated mice are extremely susceptible to C. difficile colonization and thus should not be in direct contact with C. difficile-infected mice28-30. If the aforementioned critical steps are recognized, successful execution of the CDI mouse model is expected. 
Modification of the dose or strain of C. difficile spores administered to the mice may alter the outcomes of this model. Increasing the spore inoculum administered to mice could increase the morbidity and mortality observed, depending on the strain of C. difficile administered. A dose response experiment, evaluating a range of several inoculum doses, is recommended if using other C. difficile strains in order to characterize the clinical course of CDI. 
This protocol uses TCCFA medium for C. difficile enumeration of spores and vegetative cells collectively. However, if distinct phases (such as germination and outgrowth) of the C. difficile lifecycle are desired, differential plating is required. Non-heat-treated contents, as described in this protocol, will yield the CFU per g content of spores and vegetative cells together. If the same GI content is heat-treated (20 min at 65 &#176;C) and then plated on TCCFA media, this will yield a CFU of spores only, since C. difficile vegetative cells are destroyed by heat. Comparison of C. difficile enumeration using these two plating techniques allows for more detailed information about the C. difficile lifecycle in vivo. 
One major limitation of this mouse model is that the results may not be reproducible in mice from different strains or vendors. Recall that disruption of the gut microbiota following cefoperazone treatment renders mice susceptible to CDI28. Variations in the initial composition of the gut microbiota between different murine strains from different vendors could alter the mouse model of CDI31. Once researchers have successfully completed this model, it is recommended that the mouse strain and vendor from where the mice are acquired stay consistent for additional studies to limit variations in the gut microbiota composition, which could alter the outcomes of the model. 
Another limitation of the protocol is that the Vero cell cytotoxicity assay does not quantitatively measure C. difficile toxin levels in vivo. Instead, epithelial (Vero) cell cytotoxicity represents a qualitative approach that measures relative cytotoxicity of C. difficile toxins present in samples. Other techniques such as qRT-PCR and immunoblotting would be helpful to determine the amount of toxin present in the samples. 
Various models of CDI are currently being used, including the Syrian hamster model, ex vivo models, and human intestinal organoid models13,14,30,32. However, the mouse model of CDI provides the increased availability of commercially-available murine reagents and assays to further examine the pathogenesis of C. difficile. Other mouse models of CDI have used different strains of C. difficile, such as VPI 10463, which results in substantial mortality and thus does not recapitulate human CDI12,17,29. In this model, C. difficile R20291 was selected for its genetic tractability and clinical relevance as a recent 027 epidemic strain33,34. Compared to other murine models of CDI, R20291 was not uniformly lethal at the dose administered, allowing for observation of a prolonged clinical course of infection concordant with human disease. Overall, this mouse model proves to be a valuable experimental platform of CDI. This fully-characterized C. difficile mouse model can be utilized to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Clostridium difficile is an anaerobic, gram-positive, spore-forming bacillus that causes life-threatening diarrhea1. C. difficile infection (CDI) is associated with increased human morbidity and mortality and results in over $ 4.8 billion in healthcare costs per year1-4. In 2013, the Centers for Disease Control and Prevention categorized C. difficile as an urgent antibiotic resistance risk, indicating that it poses an urgent threat to public health1. Currently, antibiotic treatment with vancomycin and metronidazole are considered the standard of care for CDI5. Unfortunately, recurrence of CDI after successful treatment with antibiotics is high, occurring in 20 - 30% of patients2,5-7. Therefore, the discovery of novel therapeutics against this enteric pathogen is necessary. To evaluate therapeutics against C. difficile, an animal model approximating the human disease in a clinically-relevant strain is needed.
Initially, Koch&#39;s postulates were established for C. difficile in 1977 using a clindamycin-treated Syrian hamster model8. This model is still utilized today to investigate the effects of C. difficile toxins on pathogenesis9,10. However, CDI in the hamster model results in high mortality rates and does not approximate the chronic insidious disease manifestations that can be seen in humans with CDI 10,11. Based on the accessibility and reagent availability of murine platforms in research, a mouse model of CDI is relevant.
In 2008, a robust mouse model of CDI was established by treating mice with an antibiotic cocktail in drinking water (kanamycin, gentamicin, colistin, metronidazole, and vancomycin) for 3 days followed by an intraperitoneal injection of clindamycin12. This rendered mice susceptible to CDI and severe colitis. Depending on the inoculum dose administered, a range of clinical signs and lethality can be observed using this model. Since this time, various antibiotic regimens have been investigated that alter the murine gut microbiota, decreasing colonization resistance to the point where C. difficile can colonize the gastrointestinal tract (reviewed in Best et al. and Lawley &#38; Young)13,14.
More recently, a broad spectrum cephalosporin, cefoperazone, given in the drinking water for 5 or 10 days reproducibly renders mice susceptible to CDI15. Since administration of third-generation cephalosporins are associated with an increased risk of CDI in humans, use of the cefoperazone model more accurately reflects naturally-occurring disease16. Cefoperazone-treated mice susceptible to C. difficile have been challenged with both C. difficile spores and vegetative cells of a variety of strains ranging in clinical relevance and virulence17. Despite some of the original studies utilizing C. difficile vegetative cells as the infectious form, C. difficile spores are considered the major mode of transmission18.
In the last decade, C. difficile R20291, a NAP1/BI/027 strain, has emerged, causing epidemics of CDI19,20. We sought to determine the clinical course of disease when cefoperazone-treated mice were challenged with the clinically-relevant and genetically-tractable C. difficile strain, R20291. This protocol details the clinical course, including weight loss, bacterial colonization, toxin cytotoxicity, and histopathological changes in the gastrointestinal tract of mice challenged with C. difficile R20291 spores. Overall, this mouse model proves to be a valuable experimental platform for CDI approximating human disease. This characterized mouse model can thus be utilized to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

<table border="0" cellpadding="0" cellspacing="0" width="1037">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">#62 Perisept Sporidicial Disinfectant Cleaner&#160;</td>
			<td style="width:143px;">SSS Navigator</td>
			<td style="width:119px;">48027</td>
			<td style="width:483px;">This product will require dilution as recommended by the manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#956;m filter</td>
			<td>Fisherbrand</td>
			<td>09-720-3</td>
			<td>Alternative to filter plate for indivdiual samples tested in the Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4% Trypan Blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% Peniciilin/Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% heat inactivated FBS</td>
			<td>Gibco</td>
			<td>16140-071</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml plastic syringe&#160;</td>
			<td>BD Medical Supplies</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 ml Micro Centrifuge Screw Cap</td>
			<td>Corning</td>
			<td>430917</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well cell culture flat bottom plate</td>
			<td>Costar Corning</td>
			<td>CL3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well filter plate</td>
			<td>Millipore</td>
			<td>MSGVS2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive Seal</td>
			<td>ThermoScientific</td>
			<td>AB-0558</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Bacto Agar</td>
			<td style="width:143px;">Becton Dickinson</td>
			<td style="width:119px;">214010</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Bacto Proteose Peptone</td>
			<td style="width:143px;">Becton Dickinson</td>
			<td style="width:119px;">211684</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefoperazone</td>
			<td>MP Bioworks</td>
			<td>199695</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefoxitine</td>
			<td>Sigma</td>
			<td>C47856</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clostridium difficile Antitoxin Kit</td>
			<td>Tech Labs</td>
			<td>T5000</td>
			<td>Used as control for Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clostridium difficile Toxin A</td>
			<td>List Biological Labs</td>
			<td>152C</td>
			<td>Positive control for Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-cycloserine</td>
			<td>Sigma</td>
			<td>C6880</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Distilled Water</td>
			<td>Gibco</td>
			<td>15230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM 1X Media</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fructose</td>
			<td>Fisher</td>
			<td>L95500</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td>Bright-Line, Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">KH2PO4</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">P285-500</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">MgSO4 (anhydrous)</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M2643</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Millex-GS 0.22 &#956;m filter</td>
			<td style="width:143px;">Millex-GS</td>
			<td style="width:119px;">SLGS033SS</td>
			<td style="width:483px;">Filter for TCCFA plates&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">Na2HPO4</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S-0876</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">S640-3</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Number 10 disposable scalpel blade</td>
			<td>Miltex, Inc</td>
			<td>4-410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Plates</td>
			<td>Fisherbrand</td>
			<td>14230244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic petri dish</td>
			<td>Kord-Valmark Brand</td>
			<td>2900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile plastic L-shaped cell spreader</td>
			<td>Fisherbrand</td>
			<td>14-665-230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Stepper</td>
			<td>Dymax Corporation</td>
			<td>T15469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurocholate</td>
			<td>Sigma</td>
			<td>T4009</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:292px;">C57BL/6J Mice</td>
			<td style="width:143px;">The Jackson Laboratory</td>
			<td align="right" style="width:119px;">664</td>
			<td style="width:483px;">Mice should be 5-8 weeks of age</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Olympus BX43F light microscope</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DP27 camera</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cellSens Dimension software&#160;</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
	</tbody>
</table>

cefoperazone-treated mouse model of clinically-relevant clostridium difficile strain r20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

During a representative study, 5-week-old C57BL/6 WT mice were pretreated with cefoperazone in their drinking water (0.5 mg/ml) for 5 days and allowed a 2-day wash out with regular drinking water. Mice were challenged with 105 spores of C. difficile R20291 via oral gavage on day 0 (Figure 1A). Mice were monitored for weight loss and clinical signs (lethargy, inappetence, diarrhea, and hunched posture) of CDI for 14 days. The challenge of C57BL/6 WT mice with C. difficile R20291 spores resulted in diarrhea within 24 hr and significant weight loss within 48 hr post-challenge (Figure 1B). Although not observed in this experiment, mice challenged with a higher dose of C. difficile R20291 can become severely ill or have greater than 20% weight loss by 48 - 72 hr post-challenge, thus necessitating humane euthanasia. Therefore, mice require a minimum of twice-daily monitoring after challenge with C. difficile spores.
In this representative study, a significant amount of weight loss and clinical signs of disease in mice were also observed on days 2 - 7 post-challenge (Figure 1B). At 7 days post-challenge, mice began to gain weight, and clinical signs of disease subsided. By the last week of the experiment, the mice appeared clinically normal, with no evidence of the clinical signs of CDI, including diarrhea.
Fecal pellets were collected prior to the challenge with C. difficile spores and every 48 hr post-challenge (Figure 1C). Prior to the antibiotic treatment, mice were not colonized with C. difficile. Within 24 hr post challenge with C. difficile spores, mice were colonized with 107 CFUs of C. difficile per g of feces (Figure 1C). Mice remained persistently colonized with C. difficile throughout the experiment, despite no evidence of weight loss or clinical signs of CDI for the last 7 days of the experiment.
<img alt="Figure 1" src="/files/ftp_upload/54850/54850fig1.jpg" /> 
Figure 1: Cefoperazone Treated Mice Challenged with C. difficile R20291 Exhibit Weight Loss and are Colonized with C. difficile. A) 5-week-old C57BL/6 WT JAX mice (n = 3M/3F per group) were pretreated with cefoperazone in their drinking water (0.5 mg/ml) for 5 days and allowed a 2-day wash out with regular drinking water. Mice were challenged with 105 spores of C. difficile R20291 via oral gavage on day 0. Mice were monitored for weight loss and clinical signs (lethargy, inappetence, diarrhea, and hunched posture) of CDI for 14 days. Feces was collected and used for bacterial enumeration prior to starting cefoperazone, immediately after finishing the antibiotic, and on days 0, 1, 2, 3, 4, 5, 7, 9, 11, 13, and 14 throughout infection. Necropsy was performed on days 0, 2, 4, 7, and 14. B) The mice lost a significant amount of their baseline weight after challenge with C. difficile R20291. The average bodyweight of mice was measured daily from day 0. Significant weight loss was seen on days 2 through 7 when compared to day 0, the baseline weight. C) C. difficile R20291 bacterial load in the feces after the challenge. Within 24 hr after the challenge with C. difficile spores, all mice were colonized with 107 CFUs (colony-forming units) per g of feces. No significant differences in these parameters were seen between females and males. Significance was determined by the non-parametric Kruskal-Wallis one-way ANOVA test followed by Dunn&#39;s posttest (*, p &#8804; 0.05; **, p &#8804; 0.01; ***, p &#8804; 0.001; ****, p &#8804; 0.0001). Error bars represent the standard deviations from the mean. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Four mice (2 males and 2 females) were humanely euthanized and prepared for necropsy on day 0, prior to infection, to serve as uninfected controls. In addition, one mouse from each cage was humanely euthanized and prepared for necropsy on days 2, 4, 7, and 14 post-challenge (Figure 1A). Enumeration of C. difficile within the cecal content was performed at each necropsy. In concordance with fecal enumeration, no C. difficile was detected in the cecal content of mice prior to the challenge with C. difficile. At 48 hr post-challenge, mice were colonized with approximately 108 CFUs of C. difficile per g of cecal content (Figure 2A). All mice remained persistently colonized throughout the experiment.
Cecal content was also assessed throughout the infection for the presence of C. difficile toxin using the Vero cell cytotoxicity assay. No evidence of C. difficile toxin cytotoxicity was detected in cecal contents prior to the challenge (Figure 2B). C. difficile cytotoxicity was detected 2 days after the challenge with C. difficile spores and persisted throughout the infection. Despite fairly uniform colonization with C. difficile, variation in the levels of C. difficile cytotoxic activity is evident in individual mice (Figure 2B).
The majority of mouse cecal content neutralized with C. difficile antitoxin during the Vero cell cytotoxicity assay and had no evidence of cytotoxicity (Vero cell rounding) in the dilutions performed. However, a few individual mice, despite retesting, had evidence of 50 - 100% cytotoxicity in the 10-1 dilution (dilution where the sample is most concentrated), with no evidence of Vero cell rounding in the other dilutions. The antitoxin utilized in this assay is a neutralizing polyclonal antibody to C. difficile toxin, prepared in goats27. Therefore, this antitoxin may not neutralize the C. difficile binary toxin that is produced by strain R20291. However, the C. difficile binary toxin is not considered to be cytotoxic10. Excessive C. difficile toxin unable to be neutralized by the antitoxin or the presence of another cytotoxic agent other than C. difficile toxin could be contributing to this finding. This observation did not affect the determination of the cytotoxicity titer for these samples, since each sample&#39;s last dilution with the 80% Vero cell rounding had no evidence of cytotoxicity when combined with the antitoxin at the specified dilution.
<img alt="Figure 2" src="/files/ftp_upload/54850/54850fig2.jpg" /> 
Figure 2: Colonization of the Cecum and Cytotoxicity during Infection with C. difficile R20291. A) Mice ceca remained persistently colonized with C. difficile throughout the experiment, despite resolution of clinical signs and observed weight gain. All mice were colonized with greater than 108 CFUs per g of cecal contents when assessed at 2 days post-challenge. B) Vero cell cytotoxicity assay from cecal content throughout infection with C. difficile R20291. Following the challenge with C. difficile spores, mice had significant levels of cytotoxicity, as measured in log10 reciprocal dilution of toxin per g of cecal content on days 2, 4, and 7 post-challenge compared to day 0 (medians represented by the line). Significance was determined by the non-parametric Kruskal-Wallis one-way ANOVA test followed by Dunn&#39;s posttest (*, p &#8804; 0.05; **, p &#8804; 0.01; ***, p &#8804; 0.001; ****, p &#8804; 0.0001). Error bars represent the standard deviations from the mean. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
It is recommended that histologic evaluation is conducted by a board-certified veterinary pathologist in a blinded manner. For tissue scoring, a 0-4 numerical scoring system should be employed to separately assess edema, inflammatory cell infiltration, and epithelial cell damage in the ileum, cecum, and colon based upon a previously-published scoring scheme17.
Upon blinded histopathologic examination of the murine ileum, cecum, and colon following challenge with C. difficile R20291, the most significant pathologic changes were noted in the cecum (Figure 3). The total cecal histologic scores were significantly different between day 0 and days 2 and 4 post-challenge. No significant lesions were noted in the ileum throughout infection (Figure 3). Milder lesions were noted in the colon. The total colonic histological scores were significantly different between day 0 and days 2 and 7 post challenge (Figure 3).
Representative H&#38;E sections of the ileum, cecum, and colon throughout infection are available in Figure 4. Each image has its respective total histological score and individual scores for epithelial damage, inflammation, and edema, as determined by a blinded board-certified veterinary pathologist (SAM).
<img alt="Figure 3" src="/files/ftp_upload/54850/54850fig3.jpg" /> 
Figure 3: Histological Scoring of the Murine Ileum, Cecum, and Colon during Infection with C. difficile R20291. Total histological scores were calculated by adding all three scores from the parameters assessed: epithelial damage, inflammation, and edema. No significant histopathological changes were noted in the ileum throughout infection. Cecal tissue contained the most significant histological lesions during CDI. The total cecal histological scores were significantly different from day 0 on days 2 and 4 post-challenge. Milder lesions were noted in the colon. The total colonic histological scores were significantly different from day 0 on days 2 and 7 post-challenge. Significance was determined by the non-parametric Kruskal-Wallis one-way ANOVA test followed by Dunn&#39;s posttest (*, p &#8804; 0.05; **, p &#8804; 0.01; ***, p &#8804; 0.001; ****, p &#8804; 0.0001). Error bars represent the standard deviations from the mean. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54850/54850fig4.jpg" /> 
Figure 4: Histopathology During Infection with C. difficile R20291. This panel contains representative H&#38;E sections of ileum, cecum, and colon throughout infection with C. difficile R20291. The total histological scores are in parentheses ( ) for the corresponding day and tissue listed: for the ileum day 0 (0), day 2 (1), day 4 (0), day 7 (0), and day 14 (0); for the cecum day 0 (0), day 2 (5), day 4 (4), day 7 (3), and day 14 (3); and for the colon day 0 (0), day 2 (4), day 4 (1), day 7 (4), and day 14 (2). Photomicrographs were obtained on a light microscope with a 5 megapixel digital camera and its accompanying software (the scale bar represents 200 &#181;m). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291 at JoVE.com

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

cefoperazone-treated-mouse-model-clinically-relevant-clostridium

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.2K Views.  North Carolina State University College of Veterinary Medicine. This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

Video: Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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 ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...