Studying the survival of the mutant zebrafish embryos during mycobacterial infection

Tuberculosis is a worldwide health issue that affects over ten million people’s lives every year. The lack of effective prevention methods and treatments makes tuberculosis a dangerous disease. Suitable animal models are needed to promote tuberculosis research. Zebrafish (Danio rerio) is a promising animal model because of its natural pathogen Mycobacterium marinum (M. marinum). Human tuberculosis is caused by Mycobacterium tuberculosis (M. tuberculosis) which is a genetically similar pathogen to M. marinum of the zebrafish. Humans and zebrafish are anatomically very different, but their mycobacterial infections share the same main features. Both pathogens infect macrophages, and the progress of the infection is almost similar in both species. This thesis studied the survival of the mutant zebrafish embryos during mycobacterial infection. Two different survival experiments, one for the AB wild type fish and another for the unc-119 homolog b (C. elegans) (unc119b) mutant fish, were made. The unc119b fish of this thesis had a mutation in their unc119b gene. The main aim of this study is to find out how the knockout of the unc119b gene affected the survival of the embryos during mycobacterial infection. Another objective of this thesis is to study only the functions of the innate immune system of the embryos against mycobacterial infection because the adaptive immune response of the embryos is not functional until four to six weeks after fertilization. The third objective is to study how the infection proceeds and appears outwards in zebrafish embryos. In this thesis, the zebrafish embryos were infected with M. marinum on the fertilization day. There were also control groups without bacterial injection. The survival of embryos/larvae was followed for 7 or 8 days. All zebrafish that showed any signs of mycobacterial infection outwards were euthanized by following the humane endpoint criteria. In the unc119b experiment, the fish were also genotyped, and the genotyping consisted of DNA extraction, polymerase chain reaction (PCR), agarose gel electrophoresis (AGE), purification of the PCR product with enzymatic treatment and sequencing. In both experiments, the fish died from M. marinum infection. In the AB wild type experiment, the infection proceeded as expected but in the unc119b mutant experiment, the fish died less than usually. Kaplan-Meier survival analyses were made for both experiments. Based on the unc119b fish survival analysis, the mutation in the unc119b gene did not affect significantly the survival of the embryos/larvae in this thesis. In both experiments, all groups infected with M. marinum had a weaker survival than the control groups without mycobacteria. The information gained from the survival experiments of this thesis can be taken advantage of in further studies. In the future, more studies are needed to study how the lack of the functional unc119b gene affects the survival of the zebrafish embryos. One purpose of this thesis is to promote the discovery of new ways to treat the global health issue, tuberculosis.