protocol
Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections Christopher Andersson1–3, Jonas Gripenland1–4 & Jörgen Johansson1–3 1Department of
Molecular Biology, Umeå University, Umeå, Sweden. 2Molecular Infection Medicine Sweden (MIMS), Umeå, Sweden. 3Umeå Center for Microbial Research (UCMR), Umeå, Sweden. 4Present address: Department of Clinical Neuroscience, Division of Neurology, Karolinska University Hospital, Stockholm, Sweden. Correspondence should be addressed to J.G. ([email protected]) or J.J. ([email protected]).
© 2015 Nature America, Inc. All rights reserved.
Published online 2 July 2015; doi:10.1038/nprot.2015.073
Microbial infections are a global health problem, particularly as microbes are continually developing resistance to antimicrobial treatments. An effective and reliable method for testing the virulence of different microbial pathogens is therefore a useful research tool. This protocol describes how the chicken embryo can be used as a trustworthy, inexpensive, ethically desirable and quickly accessible model to assess the virulence of the human bacterial pathogen Listeria monocytogenes, which can also be extended to other microbial pathogens. We provide a step-by-step protocol and figures and videos detailing the method, including egg handling, infection strategies, pathogenicity screening and isolation of infected organs. From the start of incubation of the fertilized eggs, the protocol takes 1 × 106 bacteria) are required for a successful infection15. A promising model for studying L. monocytogenes is the greater wax moth Galleria melonella16. Other in vivo models for studying L. monocytogenes infection include Drosophila melanogaster, Caenorhabditis elegans and the zebrafish (Danio rerio)17–19. These latter models have been suggested for initial screening of various strains, but their temperature maximum (≤30 °C), which is fairly distant from the temperature of a human infection and activation of virulence gene expression (37 °C), makes them less suitable. nature protocols | VOL.10 NO.8 | 2015 | 1155
protocol Table 1 | Adjustments required to adapt the protocol for other microbial pathogens.
Microbe Francisella spp.
Microbial growth conditions Tryptic soy medium supplemented with 0.1% (wt/vol) l-cysteine
Age of chicken embryo at infection 7d
Tryptic soy broth
© 2015 Nature America, Inc. All rights reserved.
Tryptic soy medium,
Mean time until death
Compartment
Infection lengthb
References
Up to 7 d Candling
Allantoic cavity
Up to 7 d Candling
24
LD100 = 1 × 104 c.f.u.d 100 µl of inoculum LD100 = 1 × 102 c.f.u.d
Mortality scored Allantoic at day 7e cavity
Up to 7 d Candling
25
100 µl of inoculum
2d Allantoic (107 inoculum) cavity
Up to 6 d Candling
Francisella novicida:
F. tularensis LVS:
23
Determination of c.f.u. by plating
~5 d F. novicida: ~5 d
LD100 = 3 to 20 × 101 c.f.u. 10 d
12 d
50 µl of inoculum
5d
Determination of c.f.u. by plating
37 °C, Overnight C. perfringens Anaerobically Reinforced clostridial medium
Screeningc
Allantoic cavity
LD100 = 4.7 × 107 c.f.u.
37 °C, Overnight E. coli
100 µl of inoculum Francisella tularensis LVS:
37 °C, OD600 = 0.9, ~8–10 h S. aureus
Infection dosea
15 d
LD100 = 1 ×
107
c.f.u.
LD50 = 1 × 105 c.f.u.
26
Determination of c.f.u. by plating
5d (105 inoculum)
37 °C, 24 h E.
tenellaf
C. albicans
Sporozoites recovered from sporocysts by mechanic and enzymatic excystation Yeast extract peptone dextrose (YPD) medium
2 × 104 f Co-infection Sporozoites with C. perfringens at day 15 10 d
8–12 d
ND
100 µl of inoculum LD100=1 ×
107
Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections Christopher Andersson1–3, Jonas Gripenland1–4 & Jörgen Johansson1–3 1Department of
Molecular Biology, Umeå University, Umeå, Sweden. 2Molecular Infection Medicine Sweden (MIMS), Umeå, Sweden. 3Umeå Center for Microbial Research (UCMR), Umeå, Sweden. 4Present address: Department of Clinical Neuroscience, Division of Neurology, Karolinska University Hospital, Stockholm, Sweden. Correspondence should be addressed to J.G. ([email protected]) or J.J. ([email protected]).
© 2015 Nature America, Inc. All rights reserved.
Published online 2 July 2015; doi:10.1038/nprot.2015.073
Microbial infections are a global health problem, particularly as microbes are continually developing resistance to antimicrobial treatments. An effective and reliable method for testing the virulence of different microbial pathogens is therefore a useful research tool. This protocol describes how the chicken embryo can be used as a trustworthy, inexpensive, ethically desirable and quickly accessible model to assess the virulence of the human bacterial pathogen Listeria monocytogenes, which can also be extended to other microbial pathogens. We provide a step-by-step protocol and figures and videos detailing the method, including egg handling, infection strategies, pathogenicity screening and isolation of infected organs. From the start of incubation of the fertilized eggs, the protocol takes 1 × 106 bacteria) are required for a successful infection15. A promising model for studying L. monocytogenes is the greater wax moth Galleria melonella16. Other in vivo models for studying L. monocytogenes infection include Drosophila melanogaster, Caenorhabditis elegans and the zebrafish (Danio rerio)17–19. These latter models have been suggested for initial screening of various strains, but their temperature maximum (≤30 °C), which is fairly distant from the temperature of a human infection and activation of virulence gene expression (37 °C), makes them less suitable. nature protocols | VOL.10 NO.8 | 2015 | 1155
protocol Table 1 | Adjustments required to adapt the protocol for other microbial pathogens.
Microbe Francisella spp.
Microbial growth conditions Tryptic soy medium supplemented with 0.1% (wt/vol) l-cysteine
Age of chicken embryo at infection 7d
Tryptic soy broth
© 2015 Nature America, Inc. All rights reserved.
Tryptic soy medium,
Mean time until death
Compartment
Infection lengthb
References
Up to 7 d Candling
Allantoic cavity
Up to 7 d Candling
24
LD100 = 1 × 104 c.f.u.d 100 µl of inoculum LD100 = 1 × 102 c.f.u.d
Mortality scored Allantoic at day 7e cavity
Up to 7 d Candling
25
100 µl of inoculum
2d Allantoic (107 inoculum) cavity
Up to 6 d Candling
Francisella novicida:
F. tularensis LVS:
23
Determination of c.f.u. by plating
~5 d F. novicida: ~5 d
LD100 = 3 to 20 × 101 c.f.u. 10 d
12 d
50 µl of inoculum
5d
Determination of c.f.u. by plating
37 °C, Overnight C. perfringens Anaerobically Reinforced clostridial medium
Screeningc
Allantoic cavity
LD100 = 4.7 × 107 c.f.u.
37 °C, Overnight E. coli
100 µl of inoculum Francisella tularensis LVS:
37 °C, OD600 = 0.9, ~8–10 h S. aureus
Infection dosea
15 d
LD100 = 1 ×
107
c.f.u.
LD50 = 1 × 105 c.f.u.
26
Determination of c.f.u. by plating
5d (105 inoculum)
37 °C, 24 h E.
tenellaf
C. albicans
Sporozoites recovered from sporocysts by mechanic and enzymatic excystation Yeast extract peptone dextrose (YPD) medium
2 × 104 f Co-infection Sporozoites with C. perfringens at day 15 10 d
8–12 d
ND
100 µl of inoculum LD100=1 ×
107
![[Infections by Listeria monocytogenes].](/assets/img/hold.png)
