Infectious Diseases

Zebrafish are, like humans, vertebrates with both an innate and adaptive immune system able to react when challenged with pathogens. The immune and inflammation responses are relatively conserved between zebrafish and its human counterpart, making it a good animal model for research on infectious diseases. In the early stages of development, zebrafish embryos lack the adaptive immune functions and thus allow researchers to focus on the innate part of the immune response. The primary (innate) response often determines the outcome of an infection. Thus, understanding the dynamics between this response and the pathogenesis of a microbe is one of the key to the discovery of drug targets.

As a result, a large range of infectious disease models have been developed using embryos, though models using adult fish also exist. Models cover a variety of bacterial, viral, parasite and fungal infections.

Present situation on infectious diseases

Over the last century, much progress was made to fight infectious diseases, with the development of several vaccines and the fast improvement of hygiene and therapies, especially with the discovery of antibiotics. However many pathogens are still nowadays accountable for far too many deaths and pose a worldwide health concern. There are 3 major reasons for this.

First the fast and increasing development of multi-resistance to available antibiotics (bacteria such as Mycobacterium tuberculosis and Staphylococcus aureus are prominent examples). Secondly, many deadly pathogens exhibit a high mutation rate of their antigens, thus hindering the immune response and the development of vaccines, such as the parasites Plasmodium falciparum and Trypanosoma brucei and viruses like HIV. At last, the recent emergence or re-emergence of pathogens (Aeromonas hydrophila, Chikungunya virus, Photorhabdus asymbiotica) means we need to understand or reconsider their pathogenesis in order to develop a sustainable therapy.

Microscopic view of cells infected by Sindbis virus.
The fungal pathogen Cryptococcus (in green) invades blood vessels (in red). Individual fungal cells are clearly visible.

Infection of zebrafish embryos

Anaesthetised zebrafish embryos are infected by a microinjection of bugs. Many injection techniques are recognised and the site will determine the type of infection. Typically, injection in the blood circulation triggers a systemic infection which will involve mainly macrophages, while injection in the muscles leads to an infection with efficient recruitment of neutrophils. Alternative methods use an enclosed organ/cavity inside the fish (such as the otic vesicle, notochord or the hindbrain ventricle) to generate a localised infection.

In vivo imaging of infection

Bacterial pathogens behave differently in vivo and in vitro, it has been shown that their mechanisms of action can change after transfer from the host to cell culture. For example, S. aureus demonstrate regular logarithmic growth in in vitro culture, although in vivo growth would be constrained by the dynamic host environment.

In research, in vivo studies aim to better characterise phagocytes and the dynamics of the host–pathogen interaction during the development of the infection. The zebrafish embryo model facilitates this goal thanks to their transparency, allowing observation of inner tissues and the pathogen in a living organism. Individual cells can be imaged at high resolution. Additionally, with the use of the genetic and chemical fluorescent tags we are able to distinguish cell types and characterise subcellular pathways.

Different types of fluorescence labelling can be used in infection models, engaging both the creation of the cell/tissue specific fish transgenic lines and the labelling of the pathogen. For example, the mpx:GFP line, with neutrophils tagged in green, is widely used in infection and immunology studies. On the other hand, bacteria can be labelled using different methods, for instance, stained with dyes reacting with their cell wall components, or transformed with plasmids carrying a fluorescence marker. These methods enable visualisation of bacteria localisation, growth, as well as disposition – important especially when two (or more) competitive strains are tested. All these tools provide a broad array of complex approaches for a better understanding of the response of immune cells during infection.

Microscopic view of the fungal pathogen Cryptococcus.
Cells infected by Sindbis virus. A relative of Chikungunya virus – are expressing the Green Fluorescent Protein, while cells expressing Interferons (molecules mediating the immune response) in response to the infection are expressing a Red Fluorescent Protein.

Compound Screening for Drug Discovery

In addition to being able to teach us about the biology of the diseases we investigate, zebrafish can also help us find new drugs to treat them. A compound (drug) can behave very differently in an animal compared to how it behaves in a laboratory setting (on Petri dish or in a plate). There can be many reasons for this, but normally, this is due to the added complexity of the living organism compared to the simplicity of the lab bench. This can cause many problems as we try to develop new medicines to help improve the treatment of infectious diseases.

The way we hope to combat this problem is by introducing the complexity of a living organism earlier in the drug discovery process [8]. Our favourite animal, the zebrafish, is perfectly placed to help us achieve this. As mentioned above the embryos of the zebrafish are transparent (see through) which means we can use the fluorescent cell lines mentioned above to see how many cells of the invading pathogen are present in the fish.

If we give some embryos an infection with fluorescent bacteria and allow that infection to grow without any drugs we can see how the disease will progress under ‘normal’ or ‘control’ conditions. Some other embryos can be infected the same way as a control fish but this time we can give them a drug to help fight the infection. By comparing the embryos which were not given any drugs to the embryos that were, we can see how well our drugs worked. This comparison is made simple by the fact we can use camera to detect the presence of the colourful bacteria in the transparent embryos.

The advantage to using the zebrafish embryo for these studies is that it is very small. Therefore we can test a large number of embryos and compounds in plates, meaning we can rapidly screen for active compounds (those which kill the infecting bacteria). The real benefit of this model is that if we find compounds which can kill the pathogen, we already know they work in a complex system. This will make continuing to develop this compound less risky as we know there is a greater likely-hood it will work in larger animals like mice and humans.

Microscopic view of Mycobacterium Marinum.
Mycobacterium Marinum. The fish equivalent of Mycobacterium tuberculosis – is expressing a red fluorescent protein. The infection spread from the hindbrain to other body sites (arrows).