The authors would like to acknowledge the technical advise from David Briles (University of Alabama at Birmingham, AL, USA). We would like to thank Yvette Chen, the Animal Welfare Officer of The University of Melbourne, and David Taylor, the Animal Facility Manager, for helpful suggestions and discussions concerning animal welfare and ethics and development of intervention criteria and humane end points for the experiments described in this manuscript.
Odilia Wijburg and Patrick Reading are supported by a NHMRC R.D. Wright Fellowship, Kirsty Short is supported by a GSK Postgraduate Support grant and a Puzey Scholarship.

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The production of this video-article was sponsored by Caliper Life Sciences.

In vivo imaging with the IVIS machine is a unique methodology that allows the real time visualization of microbial pathogenesis 4, 6-9. Here, we show the application of this technology to understanding the synergism between S. pneumoniae and influenza virus in infant mice. Due to the non-invasive nature of this technique, we have been able to monitor the progression of infection in each individual mouse over time. This has enabled us to document the kinetics of pneumococcal transmission amongst co-housed infant mice 3. The non-invasive nature of this technique enables researchers to use fewer animals per experiment and hence reduce animal usage. It is also possible to use the IVIS machine to visualize the dissemination of the infection to previously unknown sites of infection in the body, as well as sites not easily sampled by dissection (such as the middle ear).
Before commencing the imaging experiments, we confirmed that colonization levels of the bioluminescent EF3030 strain were comparable to colonization levels of the parent EF3030 strain by enumerating viable count in tissue homogenates. In addition, we first confirmed that infection with influenza virus resulted in increased load of bioluminescent EF3030 in the nasopharynx of colonized animals as demonstrated with the parent EF3030 strain (results not shown).
While the IVIS technology is easy to operate and generates reproducible and easy to understand data, experiments must be carefully designed so that the sensitivity and utility of the technology is maximised. For example, the sensitivity of the IVIS camera is affected by the depth and opacity of the tissue 8, which in our experience, increases the detection limit for bioluminescent signals originating from the lungs. In addition, dark fur and pigmented skin reduces light transmission by as much as 10-fold (compared to hairless mice) 8. While developing this method, we have optimized this protocol for use with 5 to 14 day old C57BL/6 mice and have since demonstrated that IVIS can be used to detect colonization of 20 day old C57BL/6 mice with bioluminescent S. pneumoniae EF3030. However, signal detection may be increased when these experiments are performed in nude or BALB/c mice. Nevertheless, the IVIS camera represents a novel approach to monitor, characterize and ultimately understand the in vivo development of pneumococcal disease following influenza A virus infection.

using bioluminescent imaging to investigate synergism between streptococcus pneumoniae and influenza a virus in infant mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Watch this Scientific Journal Video about Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice at JoVE.com

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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JoVE Journal

Immunology and Infection

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