Replacing rodent models for investigating the influence of the microbiome upon innate immune responses and resistance to pathogens

Aims

This project aims to replace the use of rodents with a zebrafish larval model, to study the influence of the microbiome on immune responses to pathogenic streptococcal infection.

Background

Zebrafish have a number of advantages for studying the influence of the microbiome on immune responses to infection. They are translucent, their gut physiology is similar to mammals, they develop a gut microbiome three days after fertilisation, and the fish pathogen Streptococcus iniae mimics much of the pathology of streptococcal infection in humans. Furthermore, new microbiotic colonisers can be introduced by immersion. As in other vertebrates, the microbiome is important in stimulating immune responses and some gut microbes are protective against particular bacterial pathogens. The development of a zebrafish larval model to study the importance of the microbiome in determining immune responses to infection could replace the use of rodents.

Research details and methods

This project will characterise the influence of the microbiota on the outcome of Streptococcal infection in a clinically-predictive model using germ-free larval zebrafish colonised with specific microbiota. Live imaging of larvae infected with fluorescent-tagged bacteria will be used to follow the dissemination of infection in individual larvae over time. Fish that are transgenic for fluorescent protein expression under neutrophil or macrophage-specific promotors will be used to visualise the recruitment and response of immune cells in response to infection, and for measurement of immune component transcription by qrt-PCR. 

We propose to replace the use of rodents with a zebrafish larval model, to study the influence of the microbiome upon immune responses to pathogenic Streptococcus infection. Zebrafish have a number of advantages: they are translucent, their gut physiology is similar to mammals, they develop a gut microbiome by three days post fertilisation and the fish pathogen, S. iniae, mimics much of the pathology found in humans. As in other vertebrates, the microbiome is important in stimulating immune responses and some gut microbes are protective against particular bacterial pathogens. Germ-free (Gf) zebrafish can be easily generated and maintained and new microbiotic colonisers can be non-invasively introduced into these Gf larvae. We will characterise the influence of the microbiota upon streptococcal infection outcome in a clinically-predictive replacement model using Gf larval zebrafish colonised with specific microbiota. We hypothesise that Gf fish will be more susceptible to incoming streptococcal infection than conventionally reared fish or Gf fish given specific probiotic replacements. Furthermore we will use live imaging of larvae infected with fluorescent-tagged bacteria to follow dissemination of infection in individual larvae with / without a specific microbiome over time. We will also use fish with fluorescent protein (FP) labeled inflammatory cells to visualise phagocytic capability and cell migration of neutrophils and macrophages to sites of streptococcal infection. Studies using fluorescent bacteria and FP immune cell larvae will allow the immune and infection status of individual fish to be measured in vivo over time and directly related to the presence and/or absence of specific protective microbiota. Some mammalian non-pathogenic bacteria from probiotics and faecal material are able to colonise zebrafish. In the long-term therefore this model is likely to indicate probiotics that can be used both to alter immune status and to combat Streptococcal infection.