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Immunology and Infection

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Published: October 8th, 2018

DOI:

10.3791/58299

1Faculty of Medicine and Life Sciences, University of Tampere, 2Oral and Maxillofacial Unit, Tampere University Hospital

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host's immune responses with qPCR.

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

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 infections4,5. In addition, the ex utero development of zebrafish embryos has made the zebrafish a popular model in developmental biology6 and toxicology

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All zebrafish experiments have been approved by the Animal Experiment Board in Finland (ESAVI/8245/04.10.07/2015). Methods are performed according to the act (497/2013) and the government decree (564/2013) on the protection of animals used for scientific or educational purposes in Finland.

1. Culturing of Mycobacterium marinum

NOTE: Since Mycobacterium marinum is a pathogen capable of causing superficial infections in humans, find out the local guidelines fo.......

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The natural fish pathogen Mycobacterium marinum infects the internal organs of the zebrafish and produces a systemic infection with histologically visible granulomas19. Adult zebrafish are infected with M. marinum by an intraperitoneal injection. The DNA and RNA are extracted, and the mycobacterial load is measured by quantitative polymerase chain reaction (qPCR) using DNA as the template. The outline of the method is shown in

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Here we describe a qPCR-based application to measure mycobacterial loads from DNA extracted from experimentally infected adult zebrafish tissues. This application is based on primers designed around the 16S-23S rRNA internal transcribed spacer sequence40. The total mycobacterial load in a fish sample is estimated using a standard curve prepared from DNA extracted from a known number of cultured mycobacteria and assuming that one bacterium has one copy of its genome at any given moment. The detecti.......

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This work has been supported by the Finnish Cultural Foundation (H.L.), Tampere Tuberculosis Foundation (H.L., L.-M.V., M.M.H., M.P.), Foundation of the Finnish Anti-Tuberculosis Association (Suomen Tuberkuloosin Vastustamisyhdistyksen Säätiö) (H.L., M.M.H., M.P.), Sigrid Jusélius Foundation (M.P.), Emil Aaltonen Foundation (M.M.H.), Jane and Aatos Erkko Foundation (M.P.) and Academy of Finland (M.P.). Leena Mäkinen, Hanna-Leena Piippo and Jenna Ilomäki are acknowledged for their technical assistance. The authors acknowledge the Tampere Zebrafish Laboratory for their service.

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Name Company Catalog Number Comments
Mycobacterium marinum American Type Culture Collection ATCC 927
Middlebrock 7H10 agar BD, Thermo Fisher Scientific 11799042
Middlebrock OADC enrichment BD, Thermo Fisher Scientific 11718173
Middlebrock 7H9 medium BD, Thermo Fisher Scientific 11753473
Middlebrock ADC enrichment BD, Thermo Fisher Scientific 11718173
Tween 80 Sigma-Aldrich P1754
Glycerol Sigma-Aldrich G5516-500ML
GENESYS20 Spectrophotometer Thermo Fisher Scientific
Phosphate buffered saline tablets (PBS) Sigma-Aldrich P4417-50TAB
Phenol red Sigma-Aldrich P3532
27G needle Henke Sass Wolf 4710004020
1 ml syringe Henke Sass Wolf 4010.200V0
Omnican 100 30G insulin needle Braun 9151133
3-aminobenzoic acid ethyl ester (pH 7.0) Sigma-Aldrich A5040
1.5 ml homogenization tube Qiagen 13119-1000
2.8 mm ceramic beads Qiagen 13114-325
Ethanol, ETAX Aa Altia
2-propanol Sigma-Aldrich 278475
Chloroform VWR 22711.290
Guanidine thiocyanate Sigma-Aldrich G9277 FW 118.2 g/mol
Sodium citrate Sigma-Aldrich 1613859 FW 294.1 g/mol
Tris (free base) Sigma-Aldrich TRIS-RO FW 121.14 g/mol
TRI reagent Molecular Research Center TR118 Guanidine thiocyanate-phenol solution
PowerLyzer24 homogenizator Qiagen
Sonicator m08 Finnsonic
Nanodrop 2000 Thermo Fisher Scientific
SENSIFAST No-ROX SYBR, Green Master Mix Bioline BIO-98005
qPCR 96-well plate BioRad HSP9601
Optically transparent film BioRad MSB1001
C1000 Thermal cycler with CFX96 real-time system BioRad
RNase AWAY Thermo Fisher Scientific 10666421 decontamination reagent eliminating RNases
DNase I Thermo Fisher Scientific EN0525
Reverse Transcription Master Mix Fluidigm 100-6298
SsoFast Eva Green master mix BioRad 172-5211

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