Larva of the greater wax moth, Galleria mellonella, is a suitable alternative host for studying virulence of fish pathogenic Vibrio anguillarum

BMC Microbiology

Andrew P. Desbois³  Brian Austin³  Jason Weeks¹  David Verner-Jeffreys²  Stuart McMillan³ 

[1] Present address: Department of Environmental Science and Technology, Cranfield University, Bedford, UK;Centre for Environment, Fisheries and Aquaculture Science, Weymouth, UK;Marine Biotechnology Research Group, Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, UK

关键词: Wax moth larvae;    Vibriosis;    Replacement, reduction and refinement (3Rs);    Atlantic salmon;    Alternative host;   

Others  :  1221658
DOI  :  10.1186/s12866-015-0466-9

 received in 2015-03-23, accepted in 2015-06-12,  发布年份 2015

【 摘 要 】

Background

Microbial diseases cause considerable economic losses in aquaculture and new infection control measures often rely on a better understanding of pathogenicity. However, disease studies performed in fish hosts often require specialist infrastructure (e.g., aquaria), adherence to strict legislation and do not permit high-throughput approaches; these reasons justify the development of alternative hosts. This study aimed to validate the use of larvae of the greater wax moth (Galleria mellonella) to investigate virulence of the important fish pathogen, Vibrio anguillarum.

Results

Using 11 wild-type isolates of V. anguillarum, these bacteria killed larvae in a dose-dependent manner and replicated inside the haemolymph, but infected larvae were rescued by antibiotic therapy. Crucially, virulence correlated significantly and positively in larva and Atlantic salmon (Salmo salar) infection models. Challenge studies with mutants knocked out for single virulence determinants confirmed conserved roles in larva and fish infections in some cases (pJM1 plasmid, rtxA), but not all (empA, flaA, flaE).

Conclusions

The G. mellonella model is simple, more ethically acceptable than experiments on vertebrates and, crucially, does not necessitate liquid systems, which reduces infrastructure requirements and biohazard risks associated with contaminated water. The G. mellonella model may aid our understanding of microbial pathogens in aquaculture and lead to the timely introduction of new effective remedies for infectious diseases, while adhering to the principles of replacement, reduction and refinement (3Rs) and considerably reducing the number of vertebrates used in such studies.

【 授权许可】

   
2015 McMillan et al.

【 图 表 】

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【 参考文献 】

• [1]Lafferty KD, Harvell CD, Conrad JM, Friedman CS, Kent ML, Kuris AM, Powell EN, Rondeau D, Saksida SM. Infectious diseases affect marine fisheries and aquaculture economics. Ann Rev Mar Sci. 2015;7:471–96.
• [2]Froquet R, Cherix N, Burr SE, Frey J, Vilches S, Tomas JM, Cosson P. Alternative host model to evaluate Aeromonas virulence. Appl Environ Microbiol. 2007;73:5657–9.
• [3]Zak O, Oreilly T. Animal models in the evaluation of antimicrobial agents. Antimicrob Agents Chemother. 1991; 35:1527-1531.
• [4]Chamilos G, Lionakis MS, Lewis RE, Kontoyiannis DP. Role of mini-host models in the study of medically important fungi. Lancet Infect Dis. 2007; 7:42-55.
• [5]Schaeck M, Van den Broeck W, Hermans K, Decostere A. Fish as research tools: Alternatives to in vivo experiments. Altern Lab Anim. 2013; 41:219-229.
• [6]Ormonde P, Horstedt P, O’Toole R, Milton DL. Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum. J Bacteriol. 2000; 182:2326-2328.
• [7]Li L, Rock JL, Nelson DR. Identification and characterization of a repeat-in-toxin gene cluster in Vibrio anguillarum. Infect Immun. 2008; 76:2620-2632.
• [8]Mou XY, Spinard EJ, Driscoll MV, Zhao WJ, Nelson DR. H-NS Is a negative regulator of the two hemolysin/cytotoxin gene clusters in Vibrio anguillarum. Infect Immun. 2013; 81:3566-3576.
• [9]Brackman G, Celen S, Hillaert U, Van Calenbergh S, Cos P, Maes L, Nelis HJ, Coenye T. Structure-activity relationship of cinnamaldehyde analogs as inhibitors of AI-2 based quorum sensing and their effect on virulence of Vibrio spp. PLoS ONE. 2011;6, e16084.
• [10]Pang MD, Lin XQ, Hu M, Li J, Lu CP, Liu YJ. Tetrahymena: An alternative model host for evaluating virulence of Aeromonas strains. PLoS ONE. 2012; 7: Article ID e48922
• [11]Defoirdt T, Bossier P, Sorgeloos P, Verstraete W. The impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and Vibrio harveyi on their virulence towards gnotobiotically cultured Artemia franciscana. Environ Microbiol. 2005; 7:1239-1247.
• [12]van Soest JJ, Stockhammer OW, Ordas A, Bloemberg GV, Spaink HP, Meijer AH. Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda. BMC Immunol. 2011; 12:58. BioMed Central Full Text
• [13]Harvie EA, Green JM, Neely MN, Huttenlocher A. Innate immune response to Streptococcus iniae infection in zebrafish larvae. Infect Immun. 2013; 81:110-121.
• [14]Desbois AP, Coote PJ. Utility of greater wax moth larva (Galleria mellonella) for evaluating the toxicity and efficacy of new antimicrobial agents. Adv Appl Microbiol. 2012; 78:25-53.
• [15]Magnadottir B. Innate immunity of fish (overview). Fish Shellfish Immunol. 2006; 20:137-151.
• [16]Jiang H, Vilcinskas A, Kanost MR. Immunity in lepidopteran insects. In: Invertebrate Immunity. Söderhäll K, editor. Landes Bioscience and Springer Science + Business Media, Austin; 2010: p.181-204.
• [17]Neumann NF, Stafford JL, Barreda D, Ainsworth AJ, Belosevic M. Antimicrobial mechanisms of fish phagocytes and their role in host defense. Dev Comp Immunol. 2001; 25:807-825.
• [18]Cytrynska M, Mak P, Zdybicka-Barabas A, Suder P, Jakubowicz T. Purification and characterization of eight peptides from Galleria mellonella immune hemolymph. Peptides. 2007; 28:533-546.
• [19]Agius C, Roberts RJ. Melano-macrophage centres and their role in fish pathology. J Fish Dis. 2003; 26:499-509.
• [20]Desbois AP, Coote PJ. Wax moth larva (Galleria mellonella): an in vivo model for assessing the efficacy of antistaphylococcal agents. J Antimicrob Chemother. 2011; 66:1785-1790.
• [21]Austin B, Alsina M, Austin DA, Blanch AR, Grimont F, Grimont PAD, Jofre J, Koblavi S, Larsen JL, Pedersen K, Tiainen T, Verdonck L, Swings J. Identification and typing of Vibrio anguillarum - A comparison of different methods. Syst Appl Microbiol. 1995;18:285–302.
• [22]Frans I, Michiels CW, Bossier P, Willems KA, Lievens B, Rediers H. Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention. J Fish Dis. 2011; 34:643-661.
• [23]Naka H, Crosa JH. Genetic determinants of virulence in the marine fish pathogen Vibrio anguillarum. Fish Pathol. 2011; 46:1-10.
• [24]Paillard C, Le Roux F, Borrego JJ. Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution. Aquat Living Resour. 2004; 17:477-498.
• [25]Pedersen K, Gram L, Austin DA, Austin B. Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production. J Appl Microbiol. 1997; 82:365-371.
• [26]Gonzalez SF, Osorio CR, Santos Y. Development of a PCR-based method for the detection of Listonella anguillarum in fish tissues and blood samples. Dis Aquat Organ. 2003; 55:109-115.
• [27]Milton DL, O’Toole R, Horstedt P, Wolf-Watz H. Flagellin A is essential for the virulence of Vibrio anguillarum. J Bacteriol. 1996; 178:1310-1319.
• [28]Rock JL, Nelson DR. Identification and characterization of a hemolysin gene cluster in Vibrio anguillarum. Infect Immun. 2006; 74:2777-2786.
• [29]McGee K, Horstedt P, Milton DL. Identification and characterization of additional flagellin genes from Vibrio anguillarum. J Bacteriol. 1996; 178:5188-5198.
• [30]Milton DL, Norqvist A, Wolf-Watz H. Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum. J Bacteriol. 1992; 174:7235-7244.
• [31]Holm S. A simple sequentially rejective multiple test procedure. Scand J Statist. 1979; 6:65-70.
• [32]Weber B, Chen C, Milton DL. Colonization of fish skin is vital for Vibrio anguillarum to cause disease. Environ Microbiol Rep. 2010; 2:133-139.
• [33]Crosa JH, Hodges LL, Schiewe MH. Curing of a plasmid is correlated with an attenuation of virulence in the marine fish pathogen Vibrio anguillarum. Infect Immun. 1980; 27:897-902.
• [34]Olsen RJ, Watkins ME, Cantu CC, Beres SB, Musser JM. Virulence of serotype M3 Group A Streptococcus strains in wax worms (Galleria mellonella larvae). Virulence. 2011; 2:111-119.
• [35]Jander G, Rahme LG, Ausubel FM. Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects. J Bacteriol. 2000; 182:3843-3845.
• [36]Denkin SM, Nelson DR. Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence. Appl Environ Microbiol. 2004; 70:4193-4204.
• [37]Wang Y, Zhang XH, Austin B. Comparative analysis of the phenotypic characteristics of high- and low-virulent strains of Edwardsiella tarda. J Fish Dis. 2010; 33:985-994.
• [38]Amparyup P, Charoensapsri W, Tassanakajon A. Two prophenoloxidases are important for the survival of Vibrio harveyi challenged shrimp Penaeus monodon. Dev Comp Immunol. 2009; 33:247-256.
• [39]Huang WB, Ren HL, Gopalakrishnan S, Xu DD, Qiao K, Wang KJ. First molecular cloning of a molluscan caspase from variously colored abalone (Haliotis diversicolor) and gene expression analysis with bacterial challenge. Fish Shellfish Immunol. 2010; 28:587-595.
• [40]Amorim-Vaz S, Delarze E, Ischer F, Sanglard D, Coste AT. Examining the virulence of Candida albicans transcription factor mutants using Galleria mellonella and mouse infection models. Front Microbiol. 2015;6:367.
• [41]Denkin SM, Nelson DR. Induction of protease activity in Vibrio anguillarum by gastrointestinal mucus. Appl Environ Microbiol. 1999; 65:3555-3560.
• [42]Norqvist A, Hagstrom A, Wolf-Watz H. Protection of rainbow trout against vibriosis and furunculosis by the use of attenuated strains of Vibrio anguillarum. Appl Environ Microbiol. 1989; 55:1400-1405.