Name: the galleria mellonella waxworm infection model for disseminated candidiasis
Uploaded: 2018-11-17T15:00:00
Description: Watch this Scientific Journal Video about The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis at JoVE.com

The authors would like to acknowledge the assistance of Pamela Washington and Leah Anderson in obtaining Galleria mellonella for use in this study.

All methods described rely on use of invertebrate hosts and do not require Institutional Animal Care and Use Committee (IACUC) approval.
1. Galleria mellonella Waxworm Larvae
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	<li>Order larvae from wholesalers and suppliers that do not introduce hormones, antibiotics, or other treatments to the larvae and which are able to ship and deliver live specimens.
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		<li>Be sure to purchase all larvae from the same supplier during the course of experimentation. Be careful when orde...

<ol>
	<li>Kauffman, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+multicenter+surveillance+study+of+funguria+in+hospitalized+patients.+The+National+Institute+for+Allergy+and+Infectious+Diseases+(NIAID)+Mycoses+Study+Group.">Prospective multicenter surveillance study of funguria in hospitalized patients. The National Institute for Allergy and Infectious Diseases (NIAID) Mycoses Study Group.</a> Clinical Infectious Diseases. 30 (1), 14-18 (2000).</li><li>Horn, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+outcomes+of+candidemia+in+2019+patients:+data+from+the+prospective+antifungal+therapy+alliance+registry.">Epidemiology and outcomes of candidemia in 2019 patients: data from the prospective antifungal therapy alliance registry.</a> Clinical Infectious Diseases. 48 (12), 1695-1703 (2009).</li><li>Pfaller, M. A., Diekema, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+invasive+candidiasis:+a+persistent+public+health+problem.">Epidemiology of invasive candidiasis: a persistent public health problem.</a> Clinical Microbiology Reviews. 20 (1), 133-163 (2007).</li><li>Sardi, J. C., Scorzoni, L., Bernardi, T., Fusco-Almeida, A. M., Mendes Giannini, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+species:+current+epidemiology,+pathogenicity,+biofilm+formation,+natural+antifungal+products+and+new+therapeutic+options.">Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options.</a> Journal of Medical Microbiology. 62, 10-24 (2013).</li><li>Segal, E., Frenkel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+in+Vivo+Models+of+Candidiasis.">Experimental in Vivo Models of Candidiasis.</a> J Fungi (Basel). 4 (1), (2018).</li><li>Conti, H. R., Huppler, A. R., Whibley, N., Gaffen, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+candidiasis.">Animal models for candidiasis.</a> Current Protocols in Immunology. 105, 11-17 (2014).</li><li>Heymann, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+siderophore+iron+transporter+of+Candida+albicans+(Sit1p/Arn1p)+mediates+uptake+of+ferrichrome-type+siderophores+and+is+required+for+epithelial+invasion.">The siderophore iron transporter of Candida albicans (Sit1p/Arn1p) mediates uptake of ferrichrome-type siderophores and is required for epithelial invasion.</a> Infection and Immunity. 70 (9), 5246-5255 (2002).</li><li>Priest, S. J., Lorenz, M. 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W., 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=Use+of+Galleria+mellonella+as+a+model+organism+to+study+Legionella+pneumophila+infection.">Use of Galleria mellonella as a model organism to study Legionella pneumophila infection.</a> Journal of Visualized Experiments. (81), e50964 (2013).</li><li>Jacobsen, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galleria+mellonella+as+a+model+host+to+study+virulence+of+Candida.">Galleria mellonella as a model host to study virulence of Candida.</a> Virulence. 5 (2), 237-239 (2014).</li><li>Tsai, C. J., Loh, J. M., Proft, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galleria+mellonella+infection+models+for+the+study+of+bacterial+diseases+and+for+antimicrobial+drug+testing.">Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing.</a> Virulence. 7 (3), 214-229 (2016).</li><li>Hirakawa, M. P., 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=Genetic+and+phenotypic+intra-species+variation+in+Candida+albicans.">Genetic and phenotypic intra-species variation in Candida albicans.</a> Genome Research. 25 (3), 413-425 (2015).</li><li>Dunn, M. J., Kinney, G. M., Washington, P. M., Berman, J., Anderson, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversification+accompanies+gene+family+expansion+of+MED2+homologs+in+Candida+albicans.">Functional diversification accompanies gene family expansion of MED2 homologs in Candida albicans.</a> PLoS Genetics. 14 (4), (2018).</li><li>Astvad, K. M. T., Meletiadis, J., Whalley, S., Arendrup, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluconazole+Pharmacokinetics+in+Galleria+mellonella+Larvae+and+Performance+Evaluation+of+a+Bioassay+Compared+to+Liquid+Chromatography-Tandem+Mass+Spectrometry+for+Hemolymph+Specimens.">Fluconazole Pharmacokinetics in Galleria mellonella Larvae and Performance Evaluation of a Bioassay Compared to Liquid Chromatography-Tandem Mass Spectrometry for Hemolymph Specimens.</a> Antimicrobial Agents and Chemotherapy. 61 (10), (2017).</li><li>Mesa-Arango, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+non-mammalian+host+Galleria+mellonella+can+be+used+to+study+the+virulence+of+the+fungal+pathogen+Candida+tropicalis+and+the+efficacy+of+antifungal+drugs+during+infection+by+this+pathogenic+yeast.">The non-mammalian host Galleria mellonella can be used to study the virulence of the fungal pathogen Candida tropicalis and the efficacy of antifungal drugs during infection by this pathogenic yeast.</a> Med Mycol. 51 (5), 461-472 (2013).</li><li>Brennan, M., Thomas, D. Y., Whiteway, M., Kavanagh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+virulence+of+Candida+albicans+mutants+in+mice+and+Galleria+mellonella+larvae.">Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae.</a> FEMS Immunology and Medical Microbiology. 34 (2), 153-157 (2002).</li><li>Fuchs, B. B., O'Brien, E., Khoury, J. B., Mylonakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+using+Galleria+mellonella+as+a+model+host+to+study+fungal+pathogenesis.">Methods for using Galleria mellonella as a model host to study fungal pathogenesis.</a> Virulence. 1 (6), 475-482 (2010).</li><li>Kavanagh, K., Fallon, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galleria+mellonella+larvae+as+models+for+studying+fungal+virulence.">Galleria mellonella larvae as models for studying fungal virulence.</a> Fungal Biology Reviews. 24 (1-2), 79-83 (2010).</li><li>Hull, C. M., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+mating+type-like+locus+in+the+asexual+pathogenic+yeast+Candida+albicans.">Identification of a mating type-like locus in the asexual pathogenic yeast Candida albicans.</a> Science. 285 (5431), 1271-1275 (1999).</li><li>Legrand, 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=Homozygosity+at+the+MTL+locus+in+clinical+strains+of+Candida+albicans:+karyotypic+rearrangements+and+tetraploid+formation.">Homozygosity at the MTL locus in clinical strains of Candida albicans: karyotypic rearrangements and tetraploid formation.</a> Molecular Microbiology. 52 (5), 1451-1462 (2004).</li><li>Lockhart, S. R., 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=In+Candida+albicans,+white-opaque+switchers+are+homozygous+for+mating+type.">In Candida albicans, white-opaque switchers are homozygous for mating type.</a> Genetics. 162 (2), 737-745 (2002).</li><li>Miller, M. G., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White-opaque+switching+in+Candida+albicans+is+controlled+by+mating-type+locus+homeodomain+proteins+and+allows+efficient+mating.">White-opaque switching in Candida albicans is controlled by mating-type locus homeodomain proteins and allows efficient mating.</a> Cell. 110 (3), 293-302 (2002).</li><li>Wu, W., Lockhart, S. R., Pujol, C., Srikantha, T., Soll, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterozygosity+of+genes+on+the+sex+chromosome+regulates+Candida+albicans+virulence.">Heterozygosity of genes on the sex chromosome regulates Candida albicans virulence.</a> Molecular Microbiology. 64 (6), 1587-1604 (2007).</li><li>Herrero, A. B., 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=KRE5+gene+null+mutant+strains+of+Candida+albicans+are+avirulent+and+have+altered+cell+wall+composition+and+hypha+formation+properties.">KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties.</a> Eukaryotic Cell. 3 (6), 1423-1432 (2004).</li><li>Hall, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mnn2+mannosyltransferase+family+modulates+mannoprotein+fibril+length,+immune+recognition+and+virulence+of+Candida+albicans.">The Mnn2 mannosyltransferase family modulates mannoprotein fibril length, immune recognition and virulence of Candida albicans.</a> PLoS Pathogens. 9 (4), 1003276 (2013).</li><li>Wojda, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+of+the+greater+wax+moth+Galleria+mellonella.">Immunity of the greater wax moth Galleria mellonella.</a> Journal of Insect Science. 24 (3), 342-357 (2017).</li><li>Bergin, D., Reeves, E. P., Renwick, J., Wientjes, F. B., Kavanagh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+production+in+Galleria+mellonella+hemocytes:+identification+of+proteins+homologous+to+the+NADPH+oxidase+complex+of+human+neutrophils.">Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils.</a> Infection and Immunity. 73 (7), 4161-4170 (2005).</li><li>Lange, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+Sequence+of+Galleria+mellonella+(Greater+Wax+Moth).">Genome Sequence of Galleria mellonella (Greater Wax Moth).</a> Genome Announcements. 6 (2), (2018).</li><li>Krappmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lightning+up+the+worm:+How+to+probe+fungal+virulence+in+an+alternative+mini-host+by+bioluminescence.">Lightning up the worm: How to probe fungal virulence in an alternative mini-host by bioluminescence.</a> Virulence. 6 (8), 727-729 (2015).</li><li>Chowdhary, A., Voss, A., Meis, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug-resistant+Candida+auris:+'new+kid+on+the+block'+in+hospital-associated+infections.">Multidrug-resistant Candida auris: 'new kid on the block' in hospital-associated infections.</a> Journal of Hospital Infection. 94 (3), 209-212 (2016).</li><li>Delarze, E., Ischer, F., Sanglard, D., Coste, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+a+Gaussia+princeps+Luciferase+reporter+system+in+Candida+albicans+for+in+vivo+detection+in+the+Galleria+mellonella+infection+model.">Adaptation of a Gaussia princeps Luciferase reporter system in Candida albicans for in vivo detection in the Galleria mellonella infection model.</a> Virulence. 6 (7), 684-693 (2015).</li></ol>

The G. mellonella waxworm model stands as an effective tool for the rapid and reproducible analysis of C. albicans virulence. This detailed protocol relies upon consistent delivery of a defined infectious dose to the same site across a batch of larvae. Infectious dose has a profound impact on G. mellonella mortality whereas use of larvae between their initial arrival and ten days following receipt produced similar results. Loss of the C. albicansMTLa allele results in ...

Candida species are common human commensals that are capable of emerging as opportunistic pathogens in severely immunocompromised and dysbiotic patients. Although many Candida species can cause disease, C. albicans is the most prevalent cause of disseminated candidiasis1,2. Systemic disease results from C. albicans accessing the bloodstream through either direct penetration of previously restrictive host barriers or introduction at surgical sites and other breaches of the body3. Candida species utilize a range of pathogenic processes to cause systemic disease within the host including filamentation, biofilm formation, immune cell evasion and escape, and iron scavenging4. In vitro approaches exist to investigate individual pathogenic mechanisms, but animal models continue to provide the best option to investigate the entirety of disease outcome5,6. Previous research has detailed many instances of promising in vitro investigations of virulence failing to reproduce in vivo7,8. Thus, animal models are still required to assess virulence in vivo. Most disease models rely on mice to serve as a surrogate for human infections despite C. albicans inability to naturally colonize murine systems as a commensal9. Invertebrate models of disseminated candidiasis include the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the waxworm Galleria mellonella, although concerns about fundamental differences in basic physiology, host body temperatures, and routes of exposure have hampered their broad acceptance10,11. 
Most recently, the G. mellonella waxworm infection model has been adopted to model pathogenicity of a wide range of bacterial and fungal pathogens12,13,14. Advantages of this model include its relatively low cost, increased throughput, ease of use, and reduced ethical concerns regarding animal beneficence compared to murine models. For researchers, this translates into increased ability to test multiple variables, stronger confidence intervals, more rapid experimentation, and bypass of animal protocols. G. mellonella has served as a platform to rapidly assess C. albicans virulence following perturbation of genes required for biofilm formation, filamentation, and gene regulation across clinical isolates11,15,16. Recent studies have incorporated investigation of antifungal efficacy using G. mellonella to assess the pharmacokinetics of drug activity and resistance under in vivo settings, which are otherwise challenging and time consuming17,18. Yet, studies of C. albicans virulence in G. mellonella have been complicated by reportedly high levels of variation within experiments and inconsistent protocols between research groups that produce differing virulence phenotypes between mice and waxworms11,13,19,20,21. Here, we outline a G. mellonella protocol to standardize C. albicans infections, increase reproducibility in virulence experiments, and demonstrate consistency with previously described studies of virulence in murine models.
Previous studies demonstrated that the C. albicans mating type-like (MTL) locus on chromosome 5 regulates cell identity and mating competence similar to Saccharomyces cerevisiae and other Ascomycete fungi22. The majority of C. albicans isolates are heterozygous at the MTL locus, encoding one of each of the MTLa&#160;and MTL&#945; alleles (MTLa/&#945;), and are consequently sterile15,23,24. Loss of one of the MTL alleles through loss of heterozygosity (LOH) or mutation leads to homozygous MTLa or MTL&#945; strains that can undergo a phenotypic switch from the sterile &#39;white&#39; state to the mating competent &#39;opaque&#39; state25. Previous work has highlighted that loss of MTL heterozygosity also reduces virulence in murine models of systemic infection across different strain backgrounds26. Here, we detail the G. mellonella model for disseminated candidiasis using a genetically similar experimental set to depict the contribution of MTL heterozygosity to virulence in G. mellonella. We show that MTL configuration influenced C. albicans pathogenicity, where MTL&#945; strains were less virulent with respect to both MTLa/&#945; and MTLa cells, similar to findings within murine infection model26.

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			<td height="21" style="height:21px;width:427px;">Galleria mellonella</td>
			<td style="width:140px;">Snackworms.com</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Buy twice as many worms as expected to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 uL, Model 1701 N SYR Cemented needle, 26G, type 2 syringe</td>
			<td>Hamilton</td>
			<td>80000</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Petri dish, 100X15 mm, 500 pack</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tube, 1.7 mL, 500 pack</td>
			<td style="width:140px;">VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:427px;">Phosphate Buffered Saline (Biotechnology grade), 500 mL</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">97062-818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Ethanol absolute, &#8805;99.5% pure, 500 mL</td>
			<td style="width:140px;">Millipore Sigma</td>
			<td>EM-EX0276-1S</td>
			<td></td>
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			<td height="25" style="height:25px;width:427px;">autoclaved ddH2O</td>
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the galleria mellonella waxworm infection model for disseminated candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

Here, we demonstrate a reproducible method for the use of G. mellonella waxworms to investigate a disseminated candidiasis model of infection using C. albicans. The appropriate storage, maintenance, and selection of larvae for infection are critical component of insuring reproducibility in G. mellonella mortality (Figure 1A). Healthy larvae that are active, have a light yellow/tan color, and lack black patches on th...

Watch this Scientific Journal Video about The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis at JoVE.com

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

This method can help answer key questions in the Candida field about strain virulence and the contributions of specific genes to these processes. The main advantages of this technique are that it avoids concerns regarding animal use, is robust, fast, cheap, and provides high numbers of animals per experiment. Generally speaking, individuals that are new to this method will likely struggle a bit because they'll have some difficulty assessing worm viability prior to the beginning of the experiment or they might have some issues with inconsistent injection of the worms.Order larvae from wholesalers and suppliers that do not introduce hormones, antibiotics, or other treatments to the larvae and which are able to ship and deliver live specimens. When the larvae arrive, check each container to confirm the health and viability of the larvae. Healthy larvae will be completely submerged beneath the bedding, moving, and possessing a yellow, light tan coloration with few larvae containing black marks or discolorations along the body.The larvae can be stored in their containers in a ventilated space at ambient temperature for up to two weeks. To prepare the yeast cells for Galleria mellonella infection, grow the C.albicans strains to be injected overnight in three milliliters of fresh yeast-peptone-dextrose broth per strain in standard culture tubes on a rotating drum at medium speed and 30 degrees Celsius. The next morning, collect the cells by centrifugation, and wash the yeast three times in five milliliters of fresh PBS per centrifugation.After the last wash, resuspend cells in one milliliter of fresh PBS and transfer the yeast into a microcentrifuge tube. Serially dilute the cells in additional PBS at one-to-100, one-to-1, 000, and one-to-100, 000 concentrations, and count the cells from the one-to-100 and one-to-1, 000 dilutions. Adjust the cells to a 2.5 times 10 to the seventh cells per milliliter of PBS concentration in a new microcentrifuge tube, and confirm the accuracy of the infectious dose by plating the appropriate volume of the one-to-100, 000 dilution to achieve 100 colony-forming units on one yeast-peptone-dextrose agar plate for each culture to be used in infection.To infect the larvae with the Candida cells, first spread a small amount of waxworm bedding into one empty, 100-by-15 centimeter sterile Petri dish per biological replicate, and sterilize a 10-microliter syringe equipped with a 26-gauge needle three times with 10 microliters of 70%ethanol and three times with 10 microliters of PBS per wash. Next, vortex the inoculation dilution of C.albicans, and load 10 microliters of cells into the syringe. Open one container of G.mellonella larvae, and use a finger to carefully turn the bedding over to uncover larvae, selecting yellow, tan larvae of a similar size that are in good health with a lack of black pigmentation on the bodies, using the middle and index fingers and the thumb to pick up one larva in a manner similar to grasping a pencil.Roll the larva onto its back so the legs are facing up, holding the full length of the body between the fingers and thumb so that the larva is unable to curl or pull away from the injection. With the other hand, rotate the syringe so the needle bevel is facing up, and slowly insert the entire length of the bevel into the body at the junction with the rearmost left leg, ensuring that the needle penetrates the body and does not simply push the body of the larva inwards. The most critical step in this procedure is injection of the waxworm in such a way that damage to the larval body is minimized and the full bolus is inoculated.Then inject the full 10 microliters of Candida inoculum, and extract the needle. Co-house up to 10 larvae injected from the same Candida culture in each 100-by-15-millimeter Petri dish following inoculation, and incubate the larva at 37 degrees Celsius for eight days. Check the dishes every 24 hours to monitor death.The mortality can initially be assessed by darkened pigmentation, the formation of black patches or bodies, and a lack of movement. To confirm mortality, use tweezers to gently roll moribund larvae onto their backs and lightly poke the larva's underside with the tweezers. Larvae that begin to pupate into moths can be included in the analysis but should be removed if they begin to molt.No significant difference in the kinetics of G.mellonella death exists between larvae infected zero or 10 days after arrival. The characteristic signs of illness arise in larvae from both ages that succumb to the infection, beginning with discoloration that gives way to black patches before a total loss of motility and eventually death. Infection with C.albicans speeds up this process of discoloration and morbidity but does not significantly alter the cascade of phenotypic markers leading to death compared to uninfected or PBS-injected larvae.Notably, the infection of G.mellonella with mating type-like homo-or heterozygotes from any of the tested clinically isolated C.albicans strains produces high rates of mortality compared to PBS-injected animals, while injections of prototrophic derivative strains attenuates the lethal effect in this model. It's important to remember to choose healthy larvae and to be gentle in handling the larvae. If multiple strains will be inoculated, take care not to let the Candida cells sit in the PBS for too long.Following this procedure, other methods like immunological profiling and Candida localization within larvae can be performed to answer additional questions about the interactions between the host and fungal pathogen during an infection. After its development, this technique paved the way for researchers to explore Candida pathogenesis using high-throughput formats. Don't forget that working with Candida albicans requires care and that standard precautions, including the wear of personal protection equipment, should always be taken while performing this procedure.

the-galleria-mellonella-waxworm-infection-model-for-disseminated

Research

JoVE Journal

Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

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

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

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