This project was supported by grants from the Biotechnology and Biological Science&#160;Research Council&#160;(BBSRC), awarded to PRL and YL (BB/P001262/1), and the National Center for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)&#160;awarded to PRL, SMN, BDR, and YL (NC/R001596/1).

NOTE: All work described below are to be carried out in a CL2 laboratory within a class 2 microbiological safety cabinet (MSC) following local health and safety guidelines.
1. Preparation of M. bovis BCG lux for Infection
<ol>
	<li>Defrost a frozen 1.2 mL glycerol (15%) stock of M. bovis BCG lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi encoding the luciferase enzy...

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Global tuberculosis report 2018. , (2018).</li><li>Colditz, G. 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=Efficacy+of+BCG+Vaccine+in+the+prevention+of+tuberculosis:+meta-analysis+of+the+published+literature.">Efficacy of BCG Vaccine in the prevention of tuberculosis: meta-analysis of the published literature.</a> Journal of the American Medical Association. 271 (9), 698-702 (1994).</li><li>Meijer, A. H., Spaink, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-pathogen+interactions+made+transparent+with+the+zebrafish+model.">Host-pathogen interactions made transparent with the zebrafish model.</a> Current Drug Targets. 12 (7), 1000-1017 (2011).</li><li>Zhan, L., Tang, J., Sun, M., Qin, C. <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+tuberculosis+in+translational+and+precision+medicine.">Animal models for tuberculosis in translational and precision medicine.</a> Frontiers in Microbiology. 8, 717 (2017).</li><li>Gumbo, T., Lenaerts, A. J., Hanna, D., Romero, K., Nuermberger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclinical+models+for+antituberculosis+drug+development:+a+landscape+analysis.">Nonclinical models for antituberculosis drug development: a landscape analysis.</a> Journal of Infectious Diseases. 211 (Suppl 3), S83-S95 (2015).</li><li>Williams, A., Orme, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+tuberculosis:+an+overview.">Animal models of tuberculosis: an overview.</a> Microbiology Spectrum. 4 (4), (2016).</li><li>Myllym&#228;ki, H., Niskanen, M., Oksanen, K. E., R&#228;met, M. <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+in+tuberculosis+research+-+where+is+the+beef?.">Animal models in tuberculosis research - where is the beef?.</a> Expert Opinion in Drug Discovery. 10 (8), 871-883 (2015).</li><li>Flynn, J. L., Gideon, H. P., Mattila, J. T., Lin, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+studies+in+non-human+primate+models+of+tuberculosis.">Immunology studies in non-human primate models of tuberculosis.</a> Immunological Reviews. 264 (1), 60-73 (2015).</li><li>Cook, S. M., McArthur, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+Galleria+mellonella+as+a+model+host+for+human+pathogens.">Developing Galleria mellonella as a model host for human pathogens.</a> Virulence. 4 (5), 350-353 (2013).</li><li>Tsai, C. J. -. Y., Loh, J. M. S., 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>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> Insect Science. 24 (3), 342-357 (2017).</li><li>Browne, N., Heelan, 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=An+analysis+of+the+structural+and+functional+similarities+of+insect+hemocytes+and+mammalian+phagocytes.">An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes.</a> Virulence. 4 (7), 597-603 (2013).</li><li>Arteaga Blanco, L. 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=Differential+cellular+immune+response+of+Galleria+mellonella+to+Actinobacillus+pleuropneumoniae.">Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae.</a> Cell and Tissue Research. 370 (1), 153-168 (2017).</li><li>L&#243;pez Hern&#225;ndez, Y., Yero, D., Pinos-Rodr&#237;guez, J. M., Gibert, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animals+devoid+of+pulmonary+system+as+infection+models+in+the+study+of+lung+bacterial+pathogens.">Animals devoid of pulmonary system as infection models in the study of lung bacterial pathogens.</a> Frontiers in Microbiology. 6, 38 (2015).</li><li>Ramarao, N., Nielsen-Leroux, C., Lereclus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Insect+Galleria+mellonella+as+a+powerful+infection+model+to+investigate+bacterial+pathogenesis.">The Insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis.</a> Journal of Visualized Experiments. (70), e4392 (2012).</li><li>Li, Y., 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=Galleria+mellonella+-+a+novel+infection+model+for+the+Mycobacterium+tuberculosis+complex.">Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex.</a> Virulence. 9 (1), 1126-1137 (2018).</li><li>Meir, M., Grosfeld, T., Barkan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+validation+of+Galleria+mellonella+as+a+novel+model+organism+to+study+Mycobacterium+abscessus+infection,+pathogenesis,+and+treatment.">Establishment and validation of Galleria mellonella as a novel model organism to study Mycobacterium abscessus infection, pathogenesis, and treatment.</a> Antimicrobial Agents and Chemotherapy. 62 (4), (2018).</li><li>Entwistle, F. M., Coote, P. J. <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+greater+wax+moth+larvae,+Galleria+mellonella,+as+a+novel+in+vivo+model+for+non-tuberculosis+mycobacteria+infections+and+antibiotic+treatments.">Evaluation of greater wax moth larvae, Galleria mellonella, as a novel in vivo model for non-tuberculosis mycobacteria infections and antibiotic treatments.</a> Journal of Medical Microbiology. 67 (4), 585-597 (2018).</li><li>Snewin, V. A., Gares, M., #211;gaora, P., Hasan, Z., Brown, I. N., Young, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+immunity+to+mycobacterial+infection+with+luciferase+reporter+constructs.">Assessment of immunity to mycobacterial infection with luciferase reporter constructs.</a> Infection and Immunity. 67 (9), 4586-4593 (1999).</li><li>Newton, S., Martineau, A., Kampmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+whole+blood+assay+to+measure+viability+of+mycobacteria,+using+reporter-gene+tagged+BCG+or+M.Tb+(BCG+lux/M.Tb+lux).">A functional whole blood assay to measure viability of mycobacteria, using reporter-gene tagged BCG or M.Tb (BCG lux/M.Tb lux).</a> Journal of Visualized Experiments. (55), (2011).</li><li>Jorj&#227;o, A. 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=From+moths+to+caterpillars:+Ideal+conditions+for+Galleria+mellonella+rearing+for+in+vivo+microbiological+studies.">From moths to caterpillars: Ideal conditions for Galleria mellonella rearing for in vivo microbiological studies.</a> Virulence. 9 (1), 383-389 (2018).</li><li>Kavanagh, K., Sheehan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Galleria+mellonella+larvae+to+identify+novel+antimicrobial+agents+against+fungal+species+of+medical+interest.">The use of Galleria mellonella larvae to identify novel antimicrobial agents against fungal species of medical interest.</a> Journal of Fungi. 4 (3), 113 (2018).</li><li>Champion, O., Titball, R., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+G.+mellonella+larvae+to+provide+reliable+and+reproducible+results+in+the+study+of+fungal+pathogens.">Standardization of G. mellonella larvae to provide reliable and reproducible results in the study of fungal pathogens.</a> Journal of Fungi. 4 (3), 108 (2018).</li><li>Wojda, I., Taszlow, P., Jakubowicz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cold+shock+on+the+immune+response+of+the+greater+wax+moth+Galleria+mellonella+after+infection+with+entomopathogenic+bacteria+Bacillus+thuringiensis.">The effect of cold shock on the immune response of the greater wax moth Galleria mellonella after infection with entomopathogenic bacteria Bacillus thuringiensis.</a> Journal of Maria Curie-Sklodowska University. 69 (2), 7-18 (2015).</li><li>Nascimento, I. P., Leite, L. C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+passaging+in+liquid+media+and+storage+on+Mycobacterium+bovis+-+BCG+growth+capacity+and+infectivity.">The effect of passaging in liquid media and storage on Mycobacterium bovis - BCG growth capacity and infectivity.</a> FEMS Microbiology Letters. 243 (1), 81-86 (2005).</li><li>De Groote, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+studies+evaluating+mouse+models+used+for+efficacy+testing+of+experimental+drugs+against+Mycobacterium+tuberculosis.">Comparative studies evaluating mouse models used for efficacy testing of experimental drugs against Mycobacterium tuberculosis.</a> Antimicrobial Agents and Chemotherapy. 55 (3), 1237-1247 (2011).</li><li>Grosset, 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=Modeling+early+bactericidal+activity+in+murine+tuberculosis+provides+insights+into+the+activity+of+isoniazid+and+pyrazinamide.">Modeling early bactericidal activity in murine tuberculosis provides insights into the activity of isoniazid and pyrazinamide.</a> Proceedings of the National Academy of Sciences U.S.A. 109 (37), 15001-15005 (2012).</li><li>Vogel, H., Altincicek, B., Gl&#246;ckner, G., Vilcinskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+transcriptome+and+immune-gene+repertoire+of+the+lepidopteran+model+host+Galleria+mellonella.">A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella.</a> BMC Genomics. 12, 308 (2011).</li></ol>

The use of G. mellonella as an infection model has been established for a number of bacterial and fungal pathogens for the study of virulence, host-pathogen interaction, and as a screen for novel therapeutics10,22. The following discussion is based on the experimental procedure for&#160;the use of G. mellonella as an infection model for the MTBC. 
The health of the na&#239;ve larvae prior to experimentation c...

Tuberculosis (TB) is a major threat to global public health with 9 million new cases per year and 1.5 million deaths1. In addition, it is estimated that one quarter of the world&#8217;s population is infected with the causative agent of the disease, Mycobacterium tuberculosis (Mtb). Amongst the infected population, 5&#8722;10% will develop active TB disease over their lifetime. Furthermore, the emergence and spread of multi-drug resistant (MDR) and extensively-drug (XDR) resistant Mtb poses a serious threat to disease control, with 123 countries reporting at least one XDR case1. Treatment of TB requires a cocktail of at least four anti-mycobacterial drugs, of which isoniazid and rifampicin are prescribed for a minimum duration of six months; treatment is often associated with complex side effects and toxicities. Protection from the only licensed vaccine against TB, Mycobacterium bovis Bacillus Calmette-Gu&#233;rin (BCG), is variable2. An incomplete understanding of the pathogenesis of TB significantly hampers the development of new therapeutic and vaccination strategies.
For decades animal infection models have been vital for TB research to understand the basic pathogenesis and host response to infection, and to evaluate novel anti-mycobacterial agents, immuno-therapeutics and new vaccine candidates3,4. However, research using animal infection models of TB is notoriously difficult as the pathogenesis and progression of TB infection are complex, and there is no single animal model that mimics the full spectrum and important features of the disease5,6. Furthermore, animal experiments are expensive, time consuming to undertake and require full ethical justification. Nevertheless, animal infection models of TB have been described in non-human primates (e.g., macaques), guinea pigs, rabbits, cattle, pigs, mice and zebrafish, with each having their limitations3,4. The murine model is the most commonly used model due to cost, availability of inbred lines, reproducibility of infection and abundance of immunological reagents. However, they do not typically form granulomas associated with areas of hypoxia that are characteristic of latent tuberculosis infection (LTBI)6. Guinea pigs are highly susceptible to Mtb infection, with pathology and early granuloma formation similar to those in humans, and are widely used in vaccine testing; yet the lack of immunological reagents hampers their use as an infection model7. Zebrafish are suitable for large-scale screening in early-stage preclinical studies due to their small size, rapid reproduction and advanced genetic tools, but are anatomically and physiologically different to humans and are only susceptible to Mycobacterium marinum infection3. The animal models most closely resembling human Mtb infection are non-human primates (e.g., the macaque), but they are expensive and have significant ethical and practical considerations which considerably limits their use8.
The insect larva of the greater wax moth or honeycomb moth, Galleria mellonella, have become increasingly popular as an infection model for a variety of bacterial and fungal pathogens9, and as a screen for novel antimicrobial drug candidates10. G. mellonella is a successful invertebrate model due to its sophisticated innate immune system (comprised of cellular and humoral defenses) that shares a high degree of structural and functional similarity to that of vertebrates11. This includes immune mechanisms such as the phagocytosis of pathogens by hemocytes (functionally similar to mammalian macrophage and neutrophils)12,13, the production and circulation of anti-microbial peptides (AMPs) and complement-like proteins within the hemolymph (analogous to mammalian blood) of G. mellonella11. Other advantages9,14,15 of G. mellonella larvae as a model include 1) their large size (20&#8722;30 mm) which allows for easy manipulation and infection, as well as the collection of tissue and hemolymph for analyses, 2) easy maintenance at 37 &#176;C, compatible for studying human pathogens, 3) precise infection by injection without the need for anesthesia, 4) efficacy of antimicrobial agents can be assessed utilizing less drug for evaluation, 5) lack of ethical constraints compared to the use of mammals, 6) large group sizes can be used compared to animal models allowing greater reproducibility, and 7) shorter times for infection experiments are required.
In a recent study, we demonstrated that G. mellonella can be used as a novel infection model for studying the pathogenesis of infection by bioluminescent M. bovis BCG lux, a genetically modified version of the vaccine strain and member of the Mtb complex (MTBC)16. While G. mellonella has previously been used as an infection model for non-tuberculous mycobacteria (NTM), mainly M. marinum and Mycobacterium abscessus17,18, studies using MTBC are limited to that of Li et al.16. Bioluminescent non-pathogenic mycobacterial strains, which can be used at containment level (CL) 2 as a surrogate for Mtb, offer the advantages of safety and practicality over pathogenic mycobacteria. Following infection with BCG lux, larvae begin to develop early granuloma-like structures, which could provide valuable insight into the role of innate immunity in the establishment of TB infection16. In addition, this simple invertebrate infection model has the potential to provide a rapid, low-cost, and reliable evaluation of TB pathogenesis incorporating controlled challenge and multiple replicates for reproducibility. Furthermore, the model has the potential to be used to screen novel anti-TB drug and vaccine candidates in early development, reducing the overall number of animals in experimentation. The ability to measure changes in host and pathogen structure, transcriptome and proteome to determine drug targets and assess mechanisms of action of novel drugs and therapeutic vaccines, are also advantageous.
Here we describe the experimental protocols for the preparation of a bioluminescent M. bovis BCG lux inoculum and G. mellonella larvae for mycobacterial infection, as well as the determination of both larval and mycobacterial survival in response to infection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5ml reaction tube (Eppendorf)</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td></td>
		</tr>
		<tr>
			<td>20, 200 and 1000 &#181;l pipette and filtered tips</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well culture plate</td>
			<td>Greiner</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr>
			<td>25 ml pipettes and pipette boy</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>3 compartment Petri dish (94/15mm)</td>
			<td>Greiner</td>
			<td>637102</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Class II saftey cabinet</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flask with vented cap (250 ml)</td>
			<td>Corning</td>
			<td>CLS40183</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (&#62;99.7%)</td>
			<td>VWR</td>
			<td>208221.321</td>
			<td></td>
		</tr>
		<tr>
			<td>Galleria mellonella (250 per pk)</td>
			<td>Livefood Direct UK</td>
			<td>W250</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5150</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogeniser (FastPrep-24 5G )</td>
			<td>MP Biomedicals</td>
			<td>116005500</td>
			<td></td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>Corning</td>
			<td>30-240CR</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer (Autolumat LB 953)</td>
			<td>Berthold</td>
			<td>34622</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer tubes</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing matrix (S, 2.0ml)</td>
			<td>MP Biomedicals</td>
			<td>116925500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro syringe (25 &#181;l, 25 ga)</td>
			<td>SGE</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H11 agar</td>
			<td>BD Bioscience</td>
			<td>283810</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 broth</td>
			<td>BD Bioscience</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook ADC enrichment</td>
			<td>BD Bioscience</td>
			<td>212352</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook OADC enrichment</td>
			<td>BD Bioscience</td>
			<td>212240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium bovis BCG lux</td>
			<td>Various</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>n-decyl aldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>D7384-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaking incubator</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P4417-100TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysorbate 80 (Tween-80)</td>
			<td>Sigma-Aldrich</td>
			<td>P8074-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Small box</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>dark vented or non-sealed box recommended</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>Short and narrow tipped/Blunt long tweezers</td>
		</tr>
		<tr>
			<td>Winterm (V1.08)</td>
			<td>Berthold</td>
			<td>n/a</td>
			<td>Program LB953.TTB</td>
		</tr>
		<tr>
			<td>Petri dish (94/15mm)</td>
			<td>Greiner</td>
			<td>633181</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper (94mm)</td>
			<td>Any supplier</td>
			<td>n/a</td>
			<td>Cut to fit</td>
		</tr>
	</tbody>
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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.

Here we present representative data that can be obtained using the G. mellonella &#8212; BCG lux infection model and highlight the benefits of G. mellonella as an infection model for members of the MTBC (Figure 1). Experimental procedures with key technical points are outlined in Figure 2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_up...

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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.

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 ...

This protocol describes the use of Galleria mellonella as an alternative infection model to study tuberculosis, reducing the number of mammalian models used in research. When compared to conventional mammalian models such as mice, Galleria mellonella is a cheaper, ethically acceptable, and more accessible model to study tuberculosis. Further optimization of this model will allow for screening of candidate antimycobacterial drug efficacy and toxicity and will contribute to the development of urgently needed novel therapeutics for tuberculosis.Galleria mellonella possesses a complex innate immune system comparable to that of mammals. The study of host-pathogen interaction may further reveal the role of innate immunity in tuberculosis. The use of Galleria mellonella as an infection model is not limited to tuberculosis and has been widely used for other bacterial and fungal pathogens over the past decade.To begin, defrost a frozen 1.2-milliliter glycerol stock of Mycobacterium bovis BCG lux, the Montreal vaccine strain transformed with the shuttle plasmid pSMT1 carrying the luxAB genes. In a labeled 250-milliliter Erlenmeyer flask, inoculate 15 milliliters of Middlebrook 7H9 broth with a defrosted 1.2-milliliter aliquot of BCG lux. Place the flask in a sealed biosafety container and incubate at 37 degrees Celsius in an orbital shaker incubator at 220 rpm for 72 hours.After incubation, prepare 1:10 dilutions of the culture in phosphate-buffered saline in luminometer tubes, vortex, and load the luminometer tubes into the luminometer. Load the substrate 1%n-decyl aldehyde in absolute ethanol into the injector platform and measure the bioluminescence. Convert the bioluminescence to colony-forming units to check the growth of BCG lux culture.Transfer the culture to a centrifuge tube and centrifuge at 2, 175 times g for 10 minutes at room temperature to pellet the cells. Discard the supernatant into an appropriate disinfectant with known mycobactericidal activity. Wash the cell pellet twice in 50 milliliters of PBS-T by centrifuging at 2, 175 times g for 10 minutes to prevent bacterial clumping.Following the final wash, decant the waste supernatant and resuspend the mycobacterial cell pellet in the required volume of PBS-T to dilute the mycobacterial suspension to the desired cell density. Then, prepare 10-fold serial dilutions of the inoculum in 24-well plates using PBS-T. Plate out 10 microliters of each dilution onto Middlebrook 7H11 agar plates in duplicate to enumerate inoculum CFU counts.Maintain the purchased instar larvae in the dark at 18 degrees Celsius upon arrival and use within one week of purchase. Identify and select healthy larvae for experimentation based on uniform cream color, appropriate size and weight, high motility, and possessing the ability to right themselves when turned over. Using blunt-end tweezers, carefully count the healthy larvae into a Petri dish lined with a layer of filter paper and store at room temperature in the dark until use.Prepare the injection platform by taping a 94-millimeter diameter circular filter paper to a flat, raised surface. Aspirate three volumes of 70%ethanol into a 25-microliter microsyringe to sterilize, discard and further rinse with three volumes of sterile PBS-T. Then, resuspend the prepared BCG lux inoculum and aspirate 10 microliters into the sterilized microsyringe.Use a separate syringe to aspirate PBS-T for a negative control. Following 10 injections, resuspend the BCG lux inoculum to ensure uniform cell suspension. Use tweezers to pick up one larva and place onto the injection platform.On the platform, flip the larva onto its back and immobilize by securing the head and tail with the tweezers. Locate the last left proleg, counting down from the head of the larva and carefully insert the tip of the needle five to six millimeters deep at a 10 to 20-degree angle to the horizontal plane. Inject the inoculum from the syringe.After each infection, transfer the infected larva into a Petri dish lined with a layer of filter paper. A single 94-millimeter Petri dish can accommodate up to 30 larvae. Store the Petri dish in a vented, dark box inside an incubator at 37 degrees Celsius with 5%carbon dioxide.Monitor the survival of the larvae every 24 hours. Larvae are considered dead when they fail to move in response to touch. Record the survival and assess statistical significance with the Mantel-Cox test.Every 24 hours, randomly select five infected larvae and use a cotton bud swab soaked in 70%ethanol to gently sterilize the larval surfaces. Place the larvae individually into two-milliliter lysing matrix tubes containing 800 microliters of sterile PBS. Then, use a homogenizer to homogenize the larvae for 60 seconds at six meters per second.Centrifuge the lysing tubes at 3, 500 times g for five seconds to remove the homogenate from the lids and carefully decant the homogenate into five sterile luminometer tubes individually. Reserve 100 microliters of homogenate in a sterile 1.5-milliliter reaction tube. Recover any remaining homogenate by washing the lysing matrix tubes with one milliliter of PBS-T and pipette into the corresponding luminometer tubes.Vortex the luminometer tubes and measure the bioluminescence of the homogenates on a luminometer. Then, prepare 10-fold serial dilutions of the reserved 100 microliters of homogenate in 24-well culture plates using PBS-T. Pipette 10 microliters of the dilution onto each Middlebrook 7H11 agar plate and use a sterile plate spreader to spread.Incubate at 37 degrees Celsius for two weeks. After that, count colony-forming units. Determine the RLU to CFU ratio of BCG lux following in vivo infection.In this experiment, BCG lux dose-dependent virulence was observed in G.mellonella larvae over a 96-hour incubation period. The lethal dose required for 50%larval mortality was determined to be one times 10 to the power of seven CFU. Control groups injected with a 10-microliter dose of PBS-T or those simply pricked simulating needle injuries did not affect larval health or lead to an increase in mortality.For larvae infected with the two times 10 to the power of seven CFU dose of BCG lux, melanization of the larval dorsal line was observed from 48 hours post infection and systemic melanization was observed from 96 hours post infection. Infection with a one times 10 to the power of seven CFU dose of BCG lux resulted in an initial decline of bioluminescence within the first 72 hours post infection. However, after 72 hours post infection, the bioluminescence of BCG lux started to plateau, indicating the establishment of persistent infection.In this particular infection system, the in vivo ratio of RLU and CFU ranged from 2:1 to 5:1 with an average of 4:1 over the 168-hour time course. During injection, remember to secure the larvae so that the risk of overpenetration is minimized. Accidental puncture of the gut could lead to increased mortality.By carrying out histopathological analysis, introduction of mycobacteria with the host cells can be visualized. Further transcriptomic analysis could reveal the interaction at the genomic level. The use of this infection model may allow for the rapid testing of novel drug candidates at an early stage in development, significantly reducing the number of animals used in drug screening.This infection model requires the use of a syringe, which can be associated with needle-stick injury. We recommend the use of our injection technique to minimize such risk.

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JoVE Journal

Immunology and Infection

11.6K visualizações.  Imperial College London. Este protocolo descreve o uso da Galleria mellonella como um modelo alternativo de infecção para estudar a tuberculose, reduzindo o número de modelos mamíferos utilizados na pesquisa. Quando comparada com modelos convencionais de mamíferos, como camundongos, a Galleria mellonella é um modelo mais barato, eticamente aceitável e mais acessível para estudar a tuberculose. Uma maior otimização desse modelo permitirá a triagem da eficácia e toxicidade de medicamentos antimicobacteriano...