Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Zebrafish (Danio rerio) is a widely used animal model in biomedical research and it is an accepted model for common vertebrate biology. The zebrafish has been adapted to many fields of research modeling human diseases and disorders ranging from cancer¹ and cardiac disease² to infection and immunological studies of several bacterial ³ and viral infections^4,5. In addition, the ex utero development of zebrafish embryos has made the zebrafish a popular model in developmental biology⁶ and toxicology^7,8.

In many fields of research, including infection biology, the optically transparent zebrafish larvae are commonly used. The first immune cells appear within 24 h post fertilization (hpf), when primitive macrophages are detected⁹. Neutrophils are the next immune cells to appear around 33 hpf¹⁰. Zebrafish larvae are thus feasible for studying the early stages of infection and the role of innate immunity in the absence of adaptive immune cells¹¹. However, the adult zebrafish with its fully functional adaptive immune system provides an additional layer of complexity for infection experiments. T cells can be detected around 3 days post fertilization¹², and B cells are able to produce functional antibodies by 4 weeks post fertilization¹³. The adult zebrafish has all the main counterparts of the mammalian innate and adaptive immune system. The main differences between the immune systems of fish and humans are found in antibody isotypes as well as in the anatomy of lymphoid tissues. The zebrafish has only three antibody classes¹⁴, whereas humans have five¹⁵. In the absence of bone marrow and lymph nodes, the primary lymphoid organs in the fish are the kidney and the thymus¹⁶ and the spleen, the kidney and the gut serve as secondary lymphoid organs¹⁷. Despite these differences, with its full immune arsenal of innate and adaptive cells, the adult zebrafish is a highly applicable, easy-to-use, non-mammalian model for host-pathogen interaction studies.

The zebrafish has lately been established as a feasible model to study tuberculosis^{18,19,20,21,22}. Tuberculosis is an airborne disease caused by Mycobacterium tuberculosis. According to the World Health Organization, tuberculosis caused1.7 million deaths in 2016 and is the leading cause of death by a single pathogen worldwide²³. Mice^24,25, rabbits²⁶ and non-human primates²⁷ are the best-known animal models in tuberculosis research but each face their limitations. The non-human primate model of M. tuberculosis infection resembles the human disease most closely, but using this model is limited due to serious ethical considerations. Other animal models are hindered by the host-specificity of M. tuberculosis that affects the disease pathology. Probably the biggest issue in modeling tuberculosis is the wide spectrum of infection and disease outcomes in the human disease: tuberculosis is a very heterogeneous disease ranging from sterilizing immunity to latent, active and reactivated infection²⁸, which can be hard to reproduce and model experimentally.

Mycobacterium marinum is a close relative of M. tuberculosis with ~3,000 orthologous proteins with 85% amino acid identity²⁹. M. marinum naturally infects zebrafish producing granulomas, the hallmarks of tuberculosis, in its internal organs^19,30. Unlike other animal models used in tuberculosis research, zebrafish produces many offspring, it requires only a limited space and importantly, it is neurophysiologically the least developed vertebrate tuberculosis model available. Additionally, the M. marinum infection causes latent infection, active disease or even sterilization of mycobacterial infection in adult zebrafish closely mimicking the spectrum of disease outcomes of human tuberculosis^19,31,32. Here, we describe methods for the experimental tuberculosis model of adult zebrafish by injecting M. marinum into the abdominal cavity and using quantitative PCR to measure the mycobacterial loads and immune responses from zebrafish tissue samples.