Reduction and refinement of murine models of bacterial infection

According to estimates by the World Health Organisation, in 2002 infectious diseases caused by bacteria, viruses and parasites killed 14.7 million people worldwide ? 26% of all deaths. In the search for better vaccines and drugs, scientists often use mice to investigate how these organisms cause disease. In such studies the first signs of disease are clinical symptoms like weight loss and ruffled fur. In this research we will develop genetic tools to make a number of infectious bacteria bioluminescent and use state of the art bioluminescence imaging (BLI) technology to refine existing mouse experiments. Using very sensitive cameras we can detect the light produced by bioluminescent bacteria from within an anaesthetised animal. Multiple imaging of the same animal over the course of an experiment allows the disease progression to be followed more accurately, while using less animals. As bioluminescence is quantifiable and related to bacterial numbers, the changing bioluminescent signal can be used to estimate whether an animal will survive or die and so allows for humane euthanasia perhaps even before the appearance of clinical symptoms. We will use this technology refine our protocols and reduce the numbers of animals in experiments.

According to WHO estimates, in 2001, infectious diseases caused 14.7 million deaths ? 26% of global mortality. Murine models play an essential role in the race to develop improved vaccines and therapeutic agents but are limited by the need to use large numbers of animals to obtain quantitative microbiological data. Bioluminescence imaging (BLI) of microorganisms during infection exploits a highly sensitive, non-toxic analytical technique based on the detection of visible light produced by luciferase-catalysed reactions. BLI allows the non-invasive detection of live cells from within intact living animals in real-time. Multiple imaging of the same animal throughout an experiment allows disease progression to be followed with extreme accuracy, while allowing each animal to act as its own control. Furthermore, when constitutively expressed, bioluminescence is related to bacterial numbers and can therefore be used for quantification of pathogen burden, allowing for humane euthanasia perhaps even before the onset of clinical symptoms. The use of BLI has resulted in important new insights into the niches exploited by pathogens during infection, challenging conventional dogma and opening new avenues for research into therapeutic agents and vaccines. In this project we wish to overcome some of the obstacles to implementing BLI for infectious diseases research by constructing a suite of vectors suitable for developing dual bioluminescent/fluorescent derivatives in a wide range of both Gram-positive and Gram-negative bacteria. Importantly, even in those institutions which do not have access to BLI equipment, the ability to measure bacterial numbers in real time by luminometry will enable researchers to ensure that animals are given the correct infectious dose, reducing variation between experiments and the risk of unexpected adverse effects. In addition, we wish to develop dual bioluminescent/fluorescent derivatives of the important human pathogen Group A Streptococcus (GAS) which causes immense human morbidity and mortality and utilise BLI to interrogate and refine existing murine models of GAS infection. GAS strains produce a range of virulence factors and an array of disease manifestations, including tonsillitis, skin infections, sepsis, necrotising fasciitis, and toxic shock syndrome. Numerous murine models of GAS infection are used; ranging from non-lethal representations of colonisation and acute infections to severe invasive disease with up to 100% mortality within 24-96 hours. BLI will aid in addressing numerous outstanding questions of GAS pathogenicity and a number of the murine models used in GAS research would greatly benefit from the implementation of more humane endpoints.