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

McMillan S, Verner-Jeffreys D, Weeks J, Austin B, Desbois AP - BMC Microbiol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Marine Biotechnology Research Group, Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, UK. stuart.mcmillan@stir.ac.uk.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Survival of groups of Galleria mellonella larvae injected with culture filtrate (C.F.); 1 × 103, 1 × 105 and 1 × 107 total viable CFU; and 5 × 107 heat-killed (H.K.) CFU of four representative wild-type Vibrio anguillarum isolates with different virulence during 120 h, namely Vib1 (a), Vib64 (b), Vib79 (c) and Vib93 (d). For clarity, the unmanipulated and uninfected control groups data are not shown. n = 20, except H.K. group where n = 10
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4477312&req=5

Fig1: Survival of groups of Galleria mellonella larvae injected with culture filtrate (C.F.); 1 × 103, 1 × 105 and 1 × 107 total viable CFU; and 5 × 107 heat-killed (H.K.) CFU of four representative wild-type Vibrio anguillarum isolates with different virulence during 120 h, namely Vib1 (a), Vib64 (b), Vib79 (c) and Vib93 (d). For clarity, the unmanipulated and uninfected control groups data are not shown. n = 20, except H.K. group where n = 10

Mentions: To assess whether G. mellonella would be a suitable model for assessing virulence of V. anguillarum, it was necessary to confirm that this bacterium established an infection in the insect. Initial challenge experiments with 11 wild-type isolates demonstrated that there was dose-dependent killing of the larvae for each strain, meaning that greater inocula caused faster and greater mortality in groups (Fig. 1). Injection of larval groups with 5 × 107 CFU heat-killed bacteria had no significant effect on survival during 120 h and typically showed fewer deaths than groups challenged with the lowest dose of live bacteria (1 x103 CFU), suggesting that larval deaths were not occurring due to toxicity of bacterial cells, and viable bacteria were required to cause mortal events (Fig. 1). Moreover, sterile culture filtrate from each isolate had no significant effect on larval survival, indicating that toxic extracellular metabolites were not being produced in vitro at sufficiently high concentrations to kill the larvae and viable bacteria were required to bring about death (Fig. 1). For all strains, treatment of infected larvae with 1 μg/g TET gave a significant increase in survival compared to infected larvae treated with PBS only, thus showing that larvae could be rescued from infection with an antibiotic that inhibited the growth of the bacterium (Fig. 2). TET treatment of uninfected larvae had no significant effect on larval survival (Fig. 2). Furthermore, each isolate of V. anguillarum showed replication inside the larvae and a group of seven isolates reached ~1 x109 CFU/mL in haemolymph at 48 h while the remaining four isolates largely plateaued at 5 x107 CFU/mL from 24 h (Fig. 3). Taken together, this evidence suggests that viable and replicating V. anguillarum cells are needed to establish a systemic infection of G. mellonella, and therefore it was next important to see whether virulence of V. anguillarum isolates correlated in fish and larva models.Fig. 1


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

McMillan S, Verner-Jeffreys D, Weeks J, Austin B, Desbois AP - BMC Microbiol. (2015)

Survival of groups of Galleria mellonella larvae injected with culture filtrate (C.F.); 1 × 103, 1 × 105 and 1 × 107 total viable CFU; and 5 × 107 heat-killed (H.K.) CFU of four representative wild-type Vibrio anguillarum isolates with different virulence during 120 h, namely Vib1 (a), Vib64 (b), Vib79 (c) and Vib93 (d). For clarity, the unmanipulated and uninfected control groups data are not shown. n = 20, except H.K. group where n = 10
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4477312&req=5

Fig1: Survival of groups of Galleria mellonella larvae injected with culture filtrate (C.F.); 1 × 103, 1 × 105 and 1 × 107 total viable CFU; and 5 × 107 heat-killed (H.K.) CFU of four representative wild-type Vibrio anguillarum isolates with different virulence during 120 h, namely Vib1 (a), Vib64 (b), Vib79 (c) and Vib93 (d). For clarity, the unmanipulated and uninfected control groups data are not shown. n = 20, except H.K. group where n = 10
Mentions: To assess whether G. mellonella would be a suitable model for assessing virulence of V. anguillarum, it was necessary to confirm that this bacterium established an infection in the insect. Initial challenge experiments with 11 wild-type isolates demonstrated that there was dose-dependent killing of the larvae for each strain, meaning that greater inocula caused faster and greater mortality in groups (Fig. 1). Injection of larval groups with 5 × 107 CFU heat-killed bacteria had no significant effect on survival during 120 h and typically showed fewer deaths than groups challenged with the lowest dose of live bacteria (1 x103 CFU), suggesting that larval deaths were not occurring due to toxicity of bacterial cells, and viable bacteria were required to cause mortal events (Fig. 1). Moreover, sterile culture filtrate from each isolate had no significant effect on larval survival, indicating that toxic extracellular metabolites were not being produced in vitro at sufficiently high concentrations to kill the larvae and viable bacteria were required to bring about death (Fig. 1). For all strains, treatment of infected larvae with 1 μg/g TET gave a significant increase in survival compared to infected larvae treated with PBS only, thus showing that larvae could be rescued from infection with an antibiotic that inhibited the growth of the bacterium (Fig. 2). TET treatment of uninfected larvae had no significant effect on larval survival (Fig. 2). Furthermore, each isolate of V. anguillarum showed replication inside the larvae and a group of seven isolates reached ~1 x109 CFU/mL in haemolymph at 48 h while the remaining four isolates largely plateaued at 5 x107 CFU/mL from 24 h (Fig. 3). Taken together, this evidence suggests that viable and replicating V. anguillarum cells are needed to establish a systemic infection of G. mellonella, and therefore it was next important to see whether virulence of V. anguillarum isolates correlated in fish and larva models.Fig. 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Marine Biotechnology Research Group, Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, UK. stuart.mcmillan@stir.ac.uk.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus