L&#39;impianto in imaging in vivo presso l&#39;Imperial College è stata finanziata dal MRC.

<ol>
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T., Frankel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+of+the+murine+pathogen+Citrobacter+rodentium.">In vivo bioluminescence imaging of the murine pathogen Citrobacter rodentium.</a> Infect Immun. 74, 5391-5396 (2006).</li><li>Wiles, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+specificity,+colonization+and+clearance+dynamics+in+vivo+following+oral+challenges+with+the+murine+pathogen+Citrobacter+rodentium.">Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium.</a> Cell Microbiol. 6, 963-972 (2004).</li><li>Wiles, S., Dougan, G., Frankel, G. <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+a+'hyperinfectious'+bacterial+state+after+passage+of+Citrobacter+rodentium+through+the+host+gastrointestinal+tract.">Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract.</a> Cell Microbiol. 7, 1163-1172 (2005).</li><li>Fanning, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bifidobacterial+surface-exopolysaccharide+facilitates+commensal-host+interaction+through+immune+modulation+and+pathogen+protection.">Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 2108-2113 (2012).</li><li>Doyle, T. C., Burns, S. M., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+for+integrated+studies+of+infection.">In vivo bioluminescence imaging for integrated studies of infection.</a> Cell Microbiol. 6, 303-317 (2004).</li><li>Collins, J. W., 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=Pre-treatment+with+Bifidobacterium+breve+UCC2003+modulates+Citrobacter+rodentium-induced+colonic+inflammation+and+organ+specificity+of+infection.">Pre-treatment with Bifidobacterium breve UCC2003 modulates Citrobacter rodentium-induced colonic inflammation and organ specificity of infection.</a> Microbiology. , 10-1099 (2012).</li><li>Contag, C. H., 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=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol. Microbiol. 18, 593-603 (1995).</li><li>Hardy, 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=Extracellular+replication+of+Listeria+monocytogenes+in+the+murine+gall+bladder.">Extracellular replication of Listeria monocytogenes in the murine gall bladder.</a> Science. 303, 851-853 (2004).</li><li>Szittner, R., Meighen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence,+expression,+and+properties+of+luciferase+coded+by+lux+genes+from+a+terrestrial+bacterium.">Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium.</a> J. Biol. Chem. 265, 16581-16587 (1990).</li><li>Andreu, N., Fletcher, T., Krishnan, N., Wiles, S., Robertson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+measurement+of+antituberculosis+drug+activity+in+vitro+and+in+macrophages+using+bioluminescence.">Rapid measurement of antituberculosis drug activity in vitro and in macrophages using bioluminescence.</a> J. Antimicrob. Chemother. 67, 404-414 (2012).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, 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=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J. Biomed. Opt. 12, 024007 (2007).</li><li>Cronin, 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=High+resolution+in+vivo+bioluminescent+imaging+for+the+study+of+bacterial+tumour+targeting.">High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.</a> PLoS One. 7, e30940 (2012).</li><li>Mundy, R., MacDonald, T. T., Dougan, G., Frankel, G., Wiles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrobacter+rodentium+of+mice+and+man.">Citrobacter rodentium of mice and man.</a> Cell Microbiol. 7, 1697-1706 (2005).</li><li>Curtis, A., Calabro, K., Galarneau, J. R., Bigio, I. J., Krucker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+skin+pigmentation+in+C57BL/6+mice+affect+optical+bioluminescence+quantitation.">Temporal variations of skin pigmentation in C57BL/6 mice affect optical bioluminescence quantitation.</a> Mol. Imaging Biol. 13, 1114-1123 (2011).</li><li>Wiles, S., Crepin, V. F., Childs, G., Frankel, G., Kerton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+biophotonic+imaging+as+a+training+aid+for+administration+of+substances+in+laboratory+rodents.">Use of biophotonic imaging as a training aid for administration of substances in laboratory rodents.</a> Lab Anim. 41, 321-328 (2007).</li><li>Crepin, V. F., 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=Dissecting+the+role+of+the+Tir:Nck+and+Tir:IRTKS/IRSp53+signalling+pathways+in+vivo.">Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo.</a> Mol. Microbiol. 75, 308-323 (2010).</li><li>Willekens, I., 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=Evaluation+of+the+radiation+dose+in+micro-CT+with+optimization+of+the+scan+protocol.">Evaluation of the radiation dose in micro-CT with optimization of the scan protocol.</a> ContrastMedia Mol. Imaging. 5, 201-207 (2010).</li><li>Boone, J. M., Velazquez, O., Cherry, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-animal+X-ray+dose+from+micro-CT.">Small-animal X-ray dose from micro-CT.</a> Mol Imaging. 3, 149-158 (2004).</li><li>Sato, F., Sasaki, S., Kawashima, N., Chino, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+effects+of+whole+or+partial+body+x-irradiation+on+mice:+life+shortening.">Late effects of whole or partial body x-irradiation on mice: life shortening.</a> Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 39, 607-615 (1981).</li><li>Kohn, H. I., Kallman, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+strain+on+acute+x-ray+lethality+in+the+mouse.+II.+Recovery+rate+studies.">The influence of strain on acute x-ray lethality in the mouse. II. Recovery rate studies.</a> Radiat. Res. 6, 329-338 (1957).</li></ol>

Produzione e libero accesso a questo articolo è sponsorizzato da PerkinElmer. Gli autori Kevin P, Francesco, Jeff Meganck e Chaincy Kuo sono dipendenti di Pinza-A PerkinElmer Company. Tutti gli esperimenti sugli animali sono stati eseguiti in conformità con le procedure scientifiche Animals Act 1986 e sono state approvate dal comitato etico locale.

Il film 4D di infezione batterica fornisce un utile strumento per visualizzare e interpretare grandi quantità di dati di immagini in multimodalità modo rapido e semplice. Questa tecnica facilita l&#39;analisi dettagliata di come una infezione diffonde attraverso un individuo mouse e può essere usato per studiare come ospite o delezione di geni batterici o particolare intervento strategie effetto carica batterica, distribuzione e localizzazione durante uno studio longitudinale 7. Questi video offrono anche strumenti didattici utili e strumento di diffusione delle informazioni al pubblico. Ci sono diversi passaggi critici in questo protocollo che potrebbero influenzare la qualità dei dati ottenuti da DLIT-μCT imaging e la possibilità di compilare un video 4D di infezione. La parte più importante di questo protocollo è l&#39;infezione di successo e omogenea di topi con C. rodentium. E &#39;essenziale che i topi utilizzati per lo studio sono tra 18-20 g, e che il bacteinoculums rial sono preparati al momento e di circa 5 x 10 9 cfu, come descritto in precedenza 2,3. Prima dell&#39;infezione dei topi, è importante controllare che l&#39;inoculo è bioluminescente usando l&#39;Spectrum TC e una volta che l&#39;inoculo è stato preparato, esso deve essere continuamente omogeneizzata prima di ogni topo è gavaged per assicurare che i topi ricevono dosi infettive simili. La formazione immagine DLIT-μCT di topi è stato ottimizzato in modo che la funzione di esposizione automatica in Living software 4.3.1 Immagine determina automaticamente i parametri di imaging ottimizzati per il segnale di essere ben al di sopra del rumore. Tuttavia, la funzione di esposizione automatica si basa su impostazioni definite dall&#39;utente ei parametri che devono essere modificati come descritto nella procedura. In caso contrario, questo si tradurrà in immagini povere, con un basso numero di fotoni raccolti che non si traduca in una progressione evidente l&#39;infezione, come sono programmate le impostazioni di fabbrica della Spectrum CT per esposizione automatica per i tumori di imaging espressionelucciola luciferasi. Ricostruzioni eseguite utilizzando 560-620 nm danno il miglior accordo tra i dati simulati e misurati e, pertanto, sono i dati più affidabili per includere nella ricostruzione. Una limitazione all&#39;uso di DLIT-μCT è che le radiazioni ionizzanti dalla scansione μCT provoca danni da radiazioni sub-letale che &#39;cumulativo in uno studio longitudinale 18. Esposizione a radiazioni sub-letale può indebolire la risposta immunitaria, causare danni al DNA e apoptosi in organi interni 19. In definitiva, i danni da radiazione sub-letali cumulativo può causare la morte se il DL 50/30 per le radiazioni ionizzanti è superato, che è tra i 5-7 Gy a seconda del ceppo del mouse e l&#39;età dei topi utilizzati 18,20,21. Anche se alcuni dei danni molecolari dalle radiazioni ionizzanti può guarire, in quanto il principio generale è quello di stimare la dose conservativamente, questo non è in genere rappresentato nella pianificazione studio. Invece, l&#39;obiettivo è quello di rimanere il più below questi limiti possibile, pur compiendo gli obiettivi di studio. Ciò è particolarmente importante in questo studio perché la normale risposta immunitaria all&#39;infezione, la frequenza delle immagini, e il fatto che transgenico, immuno-composto, o fortemente infettati animali può essere più suscettibile a radiazioni ionizzanti. Pianificare l&#39;esperimento per generare un film 4D di infezione, è importante considerare la durata dell&#39;esperimento, il numero di scansioni μCT richiesto durante questo periodo e la DL 50/30 per la radiazione ionizzante per il ceppo mouse utilizzato. Un altro limite potenziale DLIT-μCT è la forza dell&#39;espressione giornalista all&#39;interno del ceppo batterico utilizzato, come questo influirà limiti di rivelazione batteriche e tempi di imaging. E &#39;altamente raccomandato che i ricercatori utilizzano convalidati ceppi batterici che sono completamente virulento, ma ottimizzato per la massima espressione operone lux, come dimostrato in precedenza per BLI2,3. Un avvertimento per il design attuale del imaging 4D è che ogni film è composto da singole scansioni DLIT-μCT che hanno diversa scalatura fotone. Questo può rendere le immagini di difficile interpretazione se le modifiche alla localizzazione del BL focolai, o la sua intensità sono sottili, o se c&#39;è un fuoco intenso BL circondato da focolai multipli debole. Pertanto, per visualizzazioni longitudinali, è importante mantenere le barre di colore coerente di tutti i punti di tempo. Il concetto di un film 4D di infezione può essere applicato a qualsiasi agente patogeno batterico opportunamente etichettato. Il futuro sviluppo di questa tecnica avrà lo scopo di utilizzare la tomografia imaging di fluorescenza (FLIT), così come DLIT per facilitare la ricerca di risposte dell&#39;ospite alle infezioni utilizzando una combinazione di agenti patogeni batterici bioluminescenti e iniettabili fluorescenti vicino i raggi infrarossi per indagare risposta dell&#39;ospite alle infezioni. , Abbiamo solo Oltre a questo in questo protocollodescrivere l&#39;uso di batteri bioluminescenti per creare 4D film di infezione. Tuttavia, in alcuni casi, può essere necessario utilizzare fluorescenti batteri etichettati, per esempio con i tag IRFP, in modo che il reporter bioluminescenza può essere usato per studiare la genetica ospitanti durante l&#39;infezione. È importante sottolineare che l&#39;uso di immagini multi-modalità che combina DLIT / FLIT-μCT ci permetterà di studiare in modo non invasivo più parametri nel corso di una infezione batterica, che contribuirà in modo significativo alla riduzione, perfezionamento, e di sostituire l&#39;uso degli animali nella ricerca scientifica come delineato nella iniziativa del NC3R ( <a href="http://www.nc3rs.org.uk/" target="_blank">http://www.nc3rs.org.uk/</a> ).

Modelli piccoli animali, in particolare quelle che utilizzano topi, sono abitualmente utilizzati per studiare la patogenesi batterica o per testare le strategie di intervento per le infezioni, come gli antibiotici, probiotici, prebiotici e vaccini 1-7. I principali letture sperimentali da infezioni animali piccoli sono carico patogeno, la localizzazione spaziale e temporale delle infezioni, e modifica la risposta immunitaria dell&#39;organismo infetto. Nel imaging ottico in vivo è uno strumento prezioso per la ricerca della malattia infettiva e può essere utilizzato per monitorare multipla letture sperimentali attraverso l&#39;uso di geni reporter (luciferasi, proteine ​​fluorescenti, beta-lattamasi, ecc), coloranti fluorescenti, nanoparticelle o sonde chemioluminescenti mirati ad un processo biologico delle proteine, o microrganismo 6. L&#39;imaging bioluminescenza (BLI) è una modalità di imaging ottico utilizzato per monitorare la colonizzazione di piccoli animali, come topi e ratti, per batterica patogenaria 3,6,8,9. I topi sono infetti da batteri ricombinanti esprimenti una luciferasi, come l&#39;operone lux CDABE da luminescens Photorhabdus. Questi batteri possono essere rilevate mediante la loro produzione luce utilizzando un CCD sede a sistema di imaging in vivo 3,6,9. È importante sottolineare che solo metabolicamente microrganismi attivi sono bioluminescente (BL), il che significa solo le cellule batteriche vitali vengono rilevati da questa metodologia 10,11. Utilizzando 2D BLI, la posizione della sorgente BL viene dedotto dalla superficie dell&#39;animale dove il segnale viene emesso 8. L&#39;esatta localizzazione anatomica del BL focolai in vivo deve essere determinato attraverso l&#39;analisi ex vivo di organi 3,6,9 In contrasto, tomografia composito 3D diffusa luce di imaging (DLIT) può essere utilizzato per compilare una ricostruzione quantitativa 3D della BL fonte 12. DLIT viene effettuata attraverso la raccolta di immagini scattate BL utilizzando filtri ottici definiti strette passa-banda esuccessivamente averli inseriti in una tomografia ottica algoritmo di ricostruzione 3D diffusa 1,7,12,13. Attualmente, l&#39;imaging multi-modalità è l&#39;unica metodologia disponibile per ottenere vera localizzazione anatomica non invasiva di bioluminescenti focolai in vivo, senza bisogno di analisi ex vivo. Recentemente, abbiamo usato una combinazione di DLIT co-registrate con μCT imaging per valutare Citrobacter rodentium (C. rodentium) dinamiche di colonizzazione dopo il trattamento profilattico con un batterio probiotico 7. C. rodentium è un patogeno enterico specifico murino utilizzato per l&#39;infezione umana modello con enteropatogeno e enterhemorrhagic Escherichia coli 14. C. infezione rodentium provoca colite, tipicamente associati con lieve perdita di peso, diarrea, polarizzazione Th1 risposta immunitaria e alterazioni patologiche distinte, tra cui colon iperplasia delle cripte e di fissaggio e cancellando lesione formazionil 14. In aggiunta a questo, C. rodentium patogenesi è stato studiato a fondo utilizzando BLI e le sue dinamiche di colonizzazione nei topi C57BL/6J sono ben documentati, rendendo questo batterio un microrganismo modello ideale per l&#39;uso con l&#39;imaging multimodale 3,4,7. Questo protocollo è il primo a delineare una metodologia per l&#39;imaging integrato DLIT-μCT di una infezione batterica utilizzando una singola piattaforma multimodalità di imaging, il IVIS Spectrum CT, e la generazione di un film 4D mostra la vera dinamica di questa infezione non invasivo.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bioluminescent C. rodentium</td>
			<td>Frankel lab</td>
			<td>ICC180</td>
			<td>Wiles et al., 2004</td>
		</tr>
		<tr>
			<td>Veet</td>
			<td>Boots</td>
			<td>&nbsp;</td>
			<td>Optimal depilation time is 7 min. Depilation works better if the cream is rubbed in well.</td>
		</tr>
		<tr>
			<td>Isofluorane (100% v/v)</td>
			<td>Abbott</td>
			<td>B506</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Medical Oxygen</td>
			<td>BOC Medical</td>
			<td>&nbsp;</td>
			<td>Size F Cylinder. Note: an appropriate regulator is required.</td>
		</tr>
		<tr>
			<td>Luria Bertani broth</td>
			<td>Merck</td>
			<td>1.10285.0500</td>
			<td>25 g in 1L Demineralised water.</td>
		</tr>
		<tr>
			<td>Luria Bertani agar</td>
			<td>Merck</td>
			<td>1.10283.0500</td>
			<td>37 g in 1L Demineralised water.</td>
		</tr>
		<tr>
			<td>Kanamycin sulphate</td>
			<td>Sigma (Fluka)</td>
			<td>60615</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml Polypropylene conical Falcon tubes</td>
			<td>BD (Falcon)</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Universals</td>
			<td>Corning (Gosselin)</td>
			<td>E5633-063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD (Plastipak)</td>
			<td>300013</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oral dosing needle (16G x 75 mm) curved</td>
			<td>Vet Tech</td>
			<td>DE005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microbanks (Cryovial)</td>
			<td>Pro-Lab Diagnostics</td>
			<td>PL.170/Y</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS Spectrum CT</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>133577 Rev A/ Spectrum CT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6kVA UPS</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>XGI-8 anesthesia system</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>118918</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>XAF-8 Anaesthesia filter charcoal</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>118999/00</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Living Image v4.3.1 SP1</td>
			<td>Caliper- a PerkinElmer Company</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benchtop shaking incubator</td>
			<td>New Brunswick Scientific</td>
			<td>Innova 44</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

4d multimodality imaging of citrobacter rodentium infections in mice

Questo protocollo descrive i passaggi necessari per monitorare longitudinalmente un bioluminescente infezione batterica utilizzando la tomografia composito 3D diffusa luce di imaging con μCT integrato (DLIT-μCT) e il successivo utilizzo di questi dati per generare un film a quattro dimensioni (4D) del ciclo di infezione. Per sviluppare il cinema 4D di infezione e di convalidare la rappresentazione DLIT-μCT per gli studi di infezione batterica utilizzando una IVIS Spectrum CT, abbiamo usato l&#39;infezione con bioluminescenti C. rodentium, che causa autolimitante colite nei topi. In questo protocollo, abbiamo delineare l&#39;infezione di topi con bioluminescenti C. rodentium e monitoraggio non invasivo di colonizzazione da imaging DLIT-μCT quotidiano e la conta batterica da feci per 8 giorni. L&#39;uso della Spectrum IVIS CT ​​facilita senza soluzione co-registrazione di scansioni ottiche e μCT utilizzando una singola piattaforma di imaging. La modalità μCT basso dosaggio permette l&#39;imaging di topi, in varie occasioni durante l&#39;infezione, fornendo dettagliata localizzazione anatomica delle foci dei batteri bioluminescenti in 3D senza causare artefatti dalla radiazione cumulativa. È importante sottolineare che il cinema 4D di topi infetti forniscono un potente strumento di analisi per monitorare le dinamiche di colonizzazione batterica in vivo.

Infezione di topi C57BL/6J con 5 x 10 9 cfu C. rodentium è un modello di infezione batterica ben descritto e si traduce in una infezione gastrointestinale autolimitante che picchi compresi tra 6-8 giorni dopo l&#39;infezione e dura da 3 a 4 settimane 2,14. L&#39;infezione è confinata al lume intestinale da parte del sistema immunitario murino e di conseguenza, i batteri sono continuamente escreto nelle feci. La colonizzazione di topi di C. rodentium può essere monitorato in modo non invasivo per enumerazione batterica diretta da feci o di imaging bioluminescenza 2,3. Questo manoscritto descrive un protocollo ottimizzato per l&#39;esecuzione di 3D e 4D multimodalità di imaging di C. rodentium all&#39;interno di topi per monitorare carica batterica e la localizzazione durante l&#39;infezione. I risultati presentati in Figura 1 dimostrano i controlli necessari per il successo di imaging 4D. Prima l&#39;infezione di topi è essenziale dissuaderemia che l&#39;inoculo batterico usato è bioluminescente (Figura 1A) e che in seguito alla sonda gastrica di un topo con bioluminescente C. rodentium, che il segnale può essere osservato nello stomaco dell&#39;animale e non nei polmoni (Figura 1B) 16. Oltre al monitoraggio colonizzazione batterica utilizzando l&#39;imaging bioluminescenza, è buona norma quantificare numeri batterici nelle feci; che viene utilizzato come una misura indiretta di colonizzazione batterica della mucosa gastrointestinale Figura 2 dimostra la tipica carica batterica nelle feci tratto da 8 C57BL6 / J. i topi che sono stati infettati con ~ 5 x 10 9 cfu ICC180 e monitorati per 8 giorni PI colonizzazione aumenta dal giorno 2 PI fino a 6-7 giorni in cui i picchi di infezione a ~ 5 x 10 10 ufc / g, che è in linea con le precedenti relazioni 2,3,17. Per sottolineare l&#39;importanza della valutazioneting la diffusione dell&#39;infezione in un singolo mouse, figure 1b e 3, così come il video 1 sono state generate dallo stesso topo seguendo C. infezione rodentium, come descritto sopra. Quotidiano DLIT-μCT stato utilizzato per valutare la distribuzione spaziale dei batteri bioluminescenti all&#39;interno di questo mouse, usare lo scheletro come un riferimento anatomico. Figura 3 dimostra la ricostruzione DLIT-μCT di un mouse C57BL/6J infettati con ICC180 a 3 punti chiave di volta PI A giorno 3 PI piccolo bioluminescente foci può essere osservato nel colon, che presentano un moderato aumento dell&#39;intensità bioluminescenza di giorno 5 PI con modeste variazioni distribuzione spaziale. Al giorno 7 PI abbiamo osservato un significativo aumento della bioluminescenza e il bioluminescente focolai sparsi in tutto il colon. Video 1 è una raccolta di ricostruzioni 3D DLIT-μCT da giorni 1-8 proporzionale con ICC180 e illustra C.rodentium all&#39;interno del tratto gastrointestinale prossimale tra i giorni 1-2 PI che si estende per i due punti al giorno 3 PI Da giorni 4-6 PI il numero di batteri presenti nel colon si espandono fino ad un massimo del 7 ° giorno PI dove i fuochi batterica copre l&#39;intero colon. Al giorno 8 PI soli due fuochi distinti batteriche sono presenti nel colon prossimale e distale. La possibilità di visualizzare la ricostruzione 3D completa in ogni punto in ordine cronologico, rappresenta un potente strumento per analizzare le interazioni patogeno ospitanti ed è più facile da interpretare che un 3D ancora dello stesso insieme di dati. <img alt="Figura 1" src="/files/ftp_upload/50450/50450fig1.jpg" /> Figura 1. Bioluminescenza immagini 2D di A) Bioluminescent C. rodentium ICC180 inoculo e B) un mouse a seguito di sonda gastrica, con 200 ml di inoculo. Punta di freccia (&gt;) illustra bioluminescenti C. rodentium </<html> em&gt; nello stomaco. <img alt="Figura 2" src="/files/ftp_upload/50450/50450fig2.jpg" /> Figura 2. C. dinamiche di colonizzazione rodentium. Quantificazione di C. rodentium unità formanti colonia di feci per 8 giorni PI <img alt="Figura 3" fo:content-width="3in" fo:src="/files/ftp_upload/50450/50450fig3highres.jpg" src="/files/ftp_upload/50450/50450fig3.jpg" /> Figura 3. Diffuse luce di imaging tomografia-μCT scansione di bioluminescenti C. infezione rodentium da un topo monitorata al giorno 3, 5 e 7 PI Arrowhead (&gt;) illustra la colonizzazione del colon. <a href="https://www.jove.com/files/ftp_upload/50450/50450fig3large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . Video 1.p :/ / www.jove.com/files/ftp_upload/50450/LAB_MEDIA_50450_Frankel_Video1.wmv &quot;target =&quot; _blank &quot;&gt; Clicca qui per vedere il video. cinema 4D di infezione da C. rodentium da un topo monitorato da giorni 1-8 PI

Watch this Scientific Journal Video about 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice at JoVE.com

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Di imaging multi-modalità è un valido approccio per studiare la colonizzazione batterica in piccoli modelli animali. Questo protocollo descrive l&#39;infezione di topi con bioluminescenti<em&gt; Citrobacter rodentium</em&gt; E il monitoraggio longitudinale della colonizzazione batterica mediante tomografia luce composito 3D diffusa con μCT di imaging per creare un filmato 4D di<em&gt; C. rodentium</em&gt; Infezione.

4D imaging multimodale di<em&gt; Citrobacter rodentium</em&gt; Infezioni in topi

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

4d-multimodality-imaging-of-citrobacter-rodentium-infections-in-mice

Research

JoVE Journal

Immunology and Infection

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

12.9K Views.  Imperial College London. Di imaging multi-modalità è un valido approccio per studiare la colonizzazione batterica in piccoli modelli animali. Questo protocollo descrive l'infezione di topi con bioluminescenti Citrobacter rodentium E il monitoraggio longitudinale della colonizzazione batterica mediante tomografia luce composito 3D diffusa con μCT di imaging per creare un filmato 4D di C. rodentium Infezione.

Video: 4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Utilizzando luciferasi di infezioni batteriche immagine in topi

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Ricerca

Immunologia e infezioni

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

Il<em&gt; Citrobacter rodentium</em&gt; Mouse Model: Studiare Contributi degli agenti patogeni e Host alla colite infettiva

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Long Term cronica<em&gt; Pseudomonas aeruginosa</em&gt; Infezione delle vie aeree nei topi

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation e Multi-fenotipica Screening-Alto contenuto di<em&gt; Coxiella burnetii</em&gt; Mutants transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Culture placentare e deciduale organo umano allo Studio infezioni all&#39;interfaccia materno-fetale

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

In Vivo Investigation of Antimicrobial Blue Light Therapy per la multi-resistente baumannii Acinetobacter Masterizzare Infezioni Utilizzando bioluminescenza Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Tubercolosi del micobatterio in topi con fluorescenza enzima Reporter di imaging

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.

Ca2 + risposte di imaging durante l'infezione delle cellule epiteliali Shigella

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Formazione immagine confocal di RNA Double-Stranded e Pattern Recognition Receptors in infezione Virus RNA negativo

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Un High-throughput Shigella-specifica analisi battericida

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...