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>
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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 ist ein anaerob, grampositive, sporenbildenden Bazillus, der lebensbedrohlichen Durchfall 1 verursacht. C. difficile - Infektion (CDI) ist mit einer erhöhten menschlichen Morbidität und Mortalität und die Ergebnisse in mehr als $ 4,8 Mrd. Kosten für das Gesundheitswesen pro Jahr 1-4 verbunden. Im Jahr 2013, kategorisiert die Centers for Disease Control and Prevention C. difficile als dringende Antibiotikaresistenz Risiko, was darauf hinweist , dass es eine dringende Gefahr für die öffentliche Gesundheit 1 darstellt. Derzeit Antibiotika - Behandlung mit Vancomycin und Metronidazol sind die Standardbehandlung für CDI 5 betrachtet. Leider ist Rezidiv von CDI nach erfolgreicher Behandlung mit Antibiotika hoch, in 20 auftretenden - 2,5-7 30% der Patienten. Deshalb ist die Entdeckung neuer Therapeutika gegen diese magensaftresistente Erreger notwendig. Zur Beurteilung Therapeutika gegen C. difficile, einem Tiermodell der Krankheit beim Menschen in ac annähertlinically relevante Belastung benötigt wird. Zunächst wurden Koch-Postulate für C. difficile im Jahr 1977 mit einer Clindamycin-behandelten syrischen Hamstermodell 8 etabliert. Dieses Modell wird auch heute noch verwendet auf Pathogenese 9,10 , die Auswirkungen von C. difficile Toxine zu untersuchen. Allerdings CDI im Hamstermodell führt zu einer hohen Sterblichkeit und nicht die chronische heimtückische Krankheit Manifestationen nähern , die bei Menschen mit CDI 10,11 gesehen werden kann. Auf der Grundlage der Zugänglichkeit und Verfügbarkeit von Reagenz murine Plattformen in Forschung, ist ein Mausmodell der CDI relevant. Im Jahr 2008 wurde mit einem Antibiotikum - Cocktail durch Behandlung von Mäusen eine robuste Mausmodell der CDI etabliert im Trinkwasser (Kanamycin, Gentamicin, Colistin, Metronidazol und Vancomycin) für 3 Tage , gefolgt von einer intraperitonealen Injektion von Clindamycin 12. Dies machte Mäuse anfällig für CDI und schwere Colitis. Abhängening auf der Inokulum Dosis verabreicht werden, können eine Reihe von klinischen Anzeichen und Letalität beobachtet werden dieses Modell. Seit dieser Zeit haben sich verschiedene Antibiotika - Anwendungen untersucht worden, die die murinen Darmmikrobiota verändern, Kolonisationsresistenz bis zu dem Punkt abnimmt , wo C. difficile den Magen - Darm - Trakt besiedeln können ( beschrieben in Best et al. Und Lawley &amp; Young) 13,14. In jüngster Zeit ein breites Spektrum Cephalosporin, Cefoperazon, rendert in dem Trinkwasser gegeben für 5 oder 10 Tage reproduzierbar Mäuse anfällig für CDI 15. Da die Verabreichung von Cephalosporinen der dritten Generation mit einem erhöhten Risiko von CDI beim Menschen assoziiert sind, reflektiert Verwendung des Cefoperazon Modell genauer natürlich vorkommenden Krankheits 16. Cefoperazon behandelten Mäuse anfällig für C. difficile wurden sowohl mit C. difficile - Sporen und vegetativen Zellen von einer Vielzahl von Stämmen Bereich in Frage gestellt worden klinischenRelevanz und Virulenz 17. Trotz einiger der ursprünglichen Studien C. difficile vegetativen Zellen als infektiöse Form verwendet werden C. difficile - Sporen die wichtigste Übertragungsart 18 betrachtet. In den letzten zehn Jahren, C. difficile R20291, ein NAP1 / BI / 027 - Stamm hat, entstanden Epidemien von CDI 19,20 verursacht. Wir versuchten , den klinischen Verlauf der Erkrankung zu bestimmen , wann Cefoperazon-behandelten Mäusen mit klinisch relevanten und genetisch tractable C. difficile - Stamm, R20291 herausgefordert wurden. Dieses Protokoll beschreibt die klinischen Verlauf, einschließlich Gewichtsverlust, bakterielle Kolonisation Toxin - Zytotoxizität und histopathologische Veränderungen im Gastrointestinaltrakt von Mäusen , die mit C. difficile - Sporen R20291 in Frage gestellt. Insgesamt erweist sich dieses Mausmodell eine wertvolle experimentelle Plattform für CDI annähert Krankheit beim Menschen zu sein. Dieses gekennzeichnet Mausmodell kann somit genutzt werden, um die Auswirkungen zu beurteilenneuartiger Therapeutika auf der Verbesserung der klinischen Erkrankung und über die Wiederherstellung der Kolonisierung Widerstand gegen C. difficile.

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	<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.

Während einer repräsentativen Studie, 5 Wochen alte C57BL / 6-WT-Mäuse wurden mit Cefoperazon in ihrem Trinkwasser vorbehandelt (0,5 mg / ml) für 5 Tage und erlaubt eine 2-Tages-auswaschen mit regelmäßigen Trinkwasser. Mäuse wurden mit 10 &amp; sup5 ; Sporen von C. difficile R20291 via Schlundsonde am Tag 0 (1A) herausgefordert. Die Mäuse wurden für 14 Tage für die Gewichtsabnahme und klinischen Zeichen (Lethargie, Appetitlosigkeit, Durchfall und gekrümmte Haltung) CDI überwacht. Die Herausforderung von C57BL / 6 - WT - Mäuse mit C. difficile R20291 Sporen führte zu Durchfall innerhalb von 24 Stunden und signifikanten Gewichtsverlust innerhalb von 48 Stunden nach der Herausforderung (Abbildung 1B). Obwohl es nicht in diesem Experiment beobachtet, mit einer höheren Dosis von C. difficile R20291 infizierte Mäuse können schwer krank geworden oder haben mehr als 20% Gewichtsverlust von 48 bis 72 Stunden nach der Herausforderung, so humane Euthanasie erforderlich macht . Deshalb erfordern Mäuse mindestens zweimal täglichÜberwachung nach mit C. difficile - Sporen Herausforderung. In dieser repräsentativen Studie, eine erhebliche Menge an Gewichtsverlust und klinischen Krankheitsanzeichen in Mäusen wurden an den Tagen beobachtet 2 - 7 post-Challenge (1B). Um 7 Tage nach der Herausforderung, Mäuse begann, Gewicht zu gewinnen, und klinischen Anzeichen einer Krankheit abgeklungen. Von der letzten Woche des Experiments schienen die Mäuse klinisch normal, ohne Anzeichen der klinischen Anzeichen von CDI, einschließlich Durchfall. Fecal Pellets wurden vor mit C. difficile - Sporen auf die Herausforderung gesammelt und alle 48 Stunden nach der Herausforderung (Abbildung 1C). Vor der Behandlung mit Antibiotika wurden Mäuse mit C. difficile nicht kolonisiert. Innerhalb von 24 Stunden nach der Herausforderung mit C. difficile - Sporen wurden die Mäuse mit 10 7 KBE von C. difficile kolonisiert pro g Kot (1C &lt;/ Strong&gt;). Mäuse blieb beharrlich mit C. difficile während des gesamten Experiments besiedelt, obwohl es keine Beweise für Gewichtsverlust oder klinischen Anzeichen von CDI für den letzten 7 Tagen des Experiments. <img alt="Abbildung 1" src="/files/ftp_upload/54850/54850fig1.jpg" /> Abbildung 1: Cefoperazon behandelten Mäuse Challenged mit C. difficile R20291 Exhibit Gewichtsverlust und sind mit C. difficile kolonisiert. A) 5 Wochen alte C57BL / 6 WT JAX - Mäuse (n = 3M / 3F pro Gruppe) wurden 5 Tage mit Cefoperazon in ihrem Trinkwasser (0,5 mg / ml) vorbehandelt und erlaubt eine 2-Tages - auswaschen mit regelmäßig zu trinken Wasser. Die Mäuse wurden mit 10 5 Sporen von C. difficile R20291 über orale Gabe am Tag herausgefordert 0 - Mäuse für Gewichtsverlust überwacht wurden , und klinische Zeichen (Lethargie, Appetitlosigkeit, Durchfall und gekrümmte Haltung) CDI für 14 Tage. Exkremente wurde für bakterielle Auszählung gesammelt und verwendet vor Cefoperazon zu starten, sofort nacr Beendigung des Antibiotikums, und an den Tagen 0, 1, 2, 3, 4, 5, 7, 9, 11, 13 und 14 im gesamten Infektion. Die Obduktion wurde an den Tagen 0 durchgeführt, 2, 4, 7 und 14 B) Die Mäuse , die eine erhebliche Menge ihres Ausgangsgewichts nach der Provokation mit C. difficile R20291 verloren. Die durchschnittliche Körpergewicht der Mäuse wurde täglich von Tag gemessen 0 signifikanten Gewichtsverlust wurde an den Tagen 2 bis 7 zu sehen, wenn sie Tag 0 verglichen, dem Basisgewicht. C) C. difficile R20291 Bakterienbelastung im Kot nach der Herausforderung. Innerhalb von 24 Stunden nach der Challenge mit C. difficile - Sporen wurden alle Mäuse mit 10 7 KBE kolonisiert (koloniebildende Einheiten) pro g der Fäzes. Keine signifikanten Unterschiede in diesen Parametern wurden zwischen Frauen und Männern gesehen. Signifikanz wurde bestimmt durch die nicht-parametrische Kruskal-Wallis - Einweg-ANOVA - Test , gefolgt von Dunn Nachtest (*, p ≤ 0,05; ** p ≤ 0,01; *** p ≤ 0.001; ****, p </em&gt; ≤ 0,0001). Fehlerbalken stellen die Standardabweichungen von dem Mittelwert. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Vier Mäuse (2 Männer und 2 Frauen) wurden auf humane Weise eingeschläfert und vorbereitet für Nekropsie am Tag 0 vor der Infektion, als nicht infizierte Kontrollen zu dienen. Darüber hinaus wurde eine Maus aus jedem Käfig auf humane Weise eingeschläfert und für Obduktion vorbereitet an den Tagen 2, 4, 7 und 14 nach dem Challenge (Abbildung 1A). Auszählung von C. difficile im Blinddarminhalt wurde bei jeder Obduktion durchgeführt. In Übereinstimmung mit fäkalen Aufzählung wurde kein C. difficile im Blinddarminhalt von Mäusen , die mit C. difficile vor der Herausforderung erkannt. Bei 48 Stunden nach der Herausforderung wurden die Mäuse kolonisiert mit etwa 10 8 KBE von C. difficile pro g Blinddarminhalt (Abbildung2A). Alle Mäuse blieben unverändert während des gesamten Experiments kolonisiert. Blinddarminhalt wurde auch während der Infektion auf das Vorhandensein von C. difficile - Toxin mit der in Vero - Zellen Zytotoxizitätstest beurteilt. Kein Nachweis von C. difficile - Toxin - Zytotoxizität wurde in cecal Inhalt vor der Herausforderung (2B) detektiert. C. difficile wurde Zytotoxizität 2 Tage nach der Herausforderung mit C. difficile - Sporen entdeckt und während der Infektion anhielt. Trotz ziemlich einheitliche Besiedlung mit C. difficile, Variation in der Höhe von C. difficile zytotoxische Aktivität ist offensichtlich in einzelnen Mäusen (2B). Die Mehrheit der Blinddarm Maus Inhalt mit C. difficile - Antitoxin während des Zell Zytotoxizitätstest Vero neutralisiert und hatte keine Hinweise auf Zytotoxizität (Vero ceRundungs ​​ll) in den durchgeführten Verdünnungen. Jedoch einige einzelne Mäuse trotz Nachtestung, zeigten Anzeichen von 50 bis 100% Zytotoxizität in der 10 -1 Verdünnung (Verdünnungs wo die Probe konzentriertesten ist), ohne Anzeichen von Vero - Zellen in den anderen Verdünnungen Rundungs. Die in diesem Assay verwendeten Antitoxin ist ein neutralisierender polyklonaler Antikörper gegen C. difficile - Toxin, hergestellt in Ziegen 27. Daher kann dieses Antitoxins nicht die C. difficile binäre Toxin neutralisieren , die durch den Stamm R20291 erzeugt wird. Jedoch ist die C. difficile binäre Toxin nicht zytotoxische 10 betrachtet werden. Übermäßige C. difficile - Toxin unfähig , durch die Antitoxin oder das Vorhandensein eines anderen zytotoxischen Mittels neutralisiert werden andere als C. difficile Toxin dieses Befundes beitragen könnte. Diese Beobachtung hatte keinen Einfluss auf die Bestimmung der Zytotoxizität Titer für diese Proben, da jeder letzten Verdünnung der Probe mit dem 80% Vero-Zell Runden hattekeine Hinweise auf Cytotoxizität, wenn sie mit dem Antitoxin bei der angegebenen Verdünnungs kombiniert. <img alt="Figur 2" src="/files/ftp_upload/54850/54850fig2.jpg" /> Abbildung 2: Die Kolonisierung des Blinddarms und Zytotoxizität während der Infektion mit C. difficile R20291. A) Mäuse ceca blieb beharrlich mit C. difficile während des gesamten Experiments kolonisiert, trotz Abklingen der klinischen Anzeichen und beobachtete Gewichtszunahme. Alle Mäuse wurden mit mehr als 10 8 KBE pro g Blinddarm Inhalt kolonisiert bei 2 Tage nach der Herausforderung bewertet. B) in Vero - Zellen Zytotoxizitätstest von Blinddarminhalt im ganzen Infektion mit C. difficile R20291. Nach dem Challenge mit C. difficile - Sporen hatten Mäuse signifikante Mengen an Cytotoxizität, wie in log 10 reziproke Verdünnung des Toxins pro g Blinddarminhalt an den Tagen 2 gemessen, 4 und 7 nach der Belastung im Vergleich zu Tag 0 (Mediane represented von der Linie). Signifikanz wurde durch den nichtparametrisches Einweg-ANOVA - Test bestimmt Kruskal-Wallis gefolgt von Dunn Nachtest (*, p ≤ 0,05; ** p ≤ 0,01; *** p ≤ 0.001; ****, p ≤ 0,0001 ). Fehlerbalken stellen die Standardabweichungen von dem Mittelwert. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig2large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> Es wird empfohlen, dass die histologische Auswertung durch einen Board-zertifizierten Veterinärpathologen in verblindet durchgeführt wird. Für Gewebe Scoring, ein 0-4 numerische Scoring - System sollte separat eingesetzt werden , um Ödeme, entzündliche Zellinfiltration und epithelialen Zellschäden im Ileum, Blinddarm und Dickdarm auf einem zuvor veröffentlichten Bewertungsschema 17 basierend beurteilen. Nach geblendet histopathologischen Prüfungnierung des murinen Ileum, Blinddarm und Dickdarm mit C. difficile R20291, die wichtigsten pathologischen Veränderungen wurden in den Blinddarm (Abbildung 3) festgestellt , nach der Herausforderung. Die Gesamt cecal histologischen Scores waren signifikant unterschiedlich zwischen Tag 0 und Tag 2 und 4 nach der Herausforderung. Keine signifikanten Läsionen wurden im Ileum gesamten Infektion (Abbildung 3) beobachtet. Milder Läsionen wurden im Dickdarm festgestellt. Die gesamten Kolon histologischen Scores waren signifikant unterschiedlich zwischen Tag 0 und 2 und 7 Tage nach der Herausforderung (Abbildung 3). Repräsentative H &amp; E Abschnitte des Ileum, Blinddarm und Kolon gesamten Infektion sind in Abbildung 4. Jedes Bild ihrer jeweiligen Gesamt histologischen Partitur und Einzelmesswerte für Epithelschädigung, Entzündungen und Ödeme hat, wie durch einen verblindeten Board-zertifizierten Veterinärpathologen bestimmt ( SAM). Abbildung 3: Histologische Bewertung des Murine Ileum, Blinddarm und Colon während der Infektion mit C. difficile R20291. Insgesamt histologischen Scores wurden durch Addition aller drei Ergebnisse aus den Parametern berechnet beurteilt: Epithelschädigung, Entzündungen und Ödeme. Keine signifikanten wurden histopathologische Veränderungen im Ileum gesamten Infektion festgestellt. Blinddarm-Gewebe enthielt die wichtigsten histologischen Veränderungen bei CDI. Die Gesamt cecal histologischen Scores unterschieden sich signifikant von Tag 0 an den Tagen 2 und 4 nach der Herausforderung. Milder Läsionen wurden im Dickdarm festgestellt. Die gesamten Kolon histologischen Scores unterschieden sich signifikant von Tag 0 an den Tagen 2 und 7 nach der Herausforderung. Signifikanz wurde bestimmt durch die nicht-parametrische Kruskal-Wallis - Einweg-ANOVA - Test , gefolgt von Dunn Nachtest (*, p ≤ 0,05; ** p ≤ 0,01; ***, p804; 0,001; ****, P ≤ 0,0001). Fehlerbalken stellen die Standardabweichungen von dem Mittelwert. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig3large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig3large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 4" src="/files/ftp_upload/54850/54850fig4.jpg" /> Abbildung 4: Histopathologie während der Infektion mit C. difficile R20291. Diese Platte enthält repräsentative H &amp; E Abschnitte von Ileum, Blinddarm und Kolon gesamten Infektion mit C. difficile R20291. Die gesamten histologischen Scores sind in Klammern () für den entsprechenden Tag und Gewebe aufgeführt: für Ileum Tag 0 (0), Tag 2 (1), Tag 4 (0), Tag 7 (0) und Tag 14 (0) ; für den Blinddarm Tag 0 (0), Tag 2 (5), Tag 4 (4), Tag 7 (3) und Tag 14 (3); und für den Darm Tag 0 (0), Tag 2 (4), Tag 4 (1), Tag 7 (4) und Tag 14 (2). Mikroskopische Aufnahmen wurden auf einem hellen microscop erhaltene mit einer 5-Megapixel-Digitalkamera und die dazugehörige Software (der Maßstabsbalken entspricht 200 &amp; mgr; m). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig4large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig4large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</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

Dieses Protokoll beschreibt die Cefoperazon Maus - Modell von Clostridium difficile - Infektion (CDI) eine klinisch relevante und genetisch-lenkbar - Stamm verwenden, R20291. Schwerpunkt auf der klinischen Krankheitsüberwachung, C. difficile Bakterien-Enumeration, Toxin Zytotoxizität und histopathologischen Veränderungen im gesamten CDI in einem Mausmodell werden im Protokoll aufgeführt.

Cefoperazon behandelten Maus-Modell der klinisch relevanten<em&gt; Clostridium difficile</em&gt; Dehnungs 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

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

12.2K Aufrufe.  North Carolina State University College of Veterinary Medicine.  Dieses Protokoll beschreibt die Cefoperazon Maus - Modell von Clostridium difficile - Infektion (CDI) eine klinisch relevante und genetisch-lenkbar - Stamm verwenden, R20291. Schwerpunkt auf der klinischen Krankheitsüberwachung, C. difficile Bakterien-Enumeration, Toxin Zytotoxizität und histopathologischen Veränderungen im gesamten CDI in einem Mausmodell werden im Protokoll aufgeführt. 

Serie: 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.

Kultivierung und Pflege<em&gt; Clostridium difficile</em&gt; In einer anaeroben Umgebung

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

Forschung

Immunologie und Infektion

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.

Prävention von Hitzestress Unerwünschte Wirkungen bei Ratten durch<em&gt; Bacillus subtilis</em&gt; Dehnungs

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.

Ein Protokoll zur Charakterisierung der morphologischen Veränderungen von<em&gt; Clostridium difficile</em&gt; In Antwort auf Antibiotika-Behandlung

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-A-Virus-Studien in einem Mausmodell der Infektion

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.

Replikation von bestellt, Nonredundant Bibliothek von Pseudomonas Aeruginosa Stamm PA14 Transposon Insertion Mutanten

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.

Ein Protein-Microarray-Assay für die serologische Bestimmung der Antigen-spezifische Antikörper Antworten folgende Clostridium Difficile Infektion

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.

Eine Reinigung und In vitro- Aktivität Assay für ein (p) PpGpp Synthestase von Clostridium difficile

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.

Eine kontrollierte Mausmodell für neonatale Sepsis Biofilm

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.

Messung der Pulsausbreitungsgeschwindigkeit, Distensibilität und Dehnung in einem Abdominal Aortenaneurysm Mausmodell

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.

Konstruktion von Mutanten im Serotyp 1 Streptococcus pneumoniae Stamm 519/43

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