Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Summary

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.

Abstract

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.

Introduction

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

Protocol

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 for personal safety and biohazard waste disposal before starting to work with this bacterium.

1. Culture M. marinum on a 7H10 plate ....

Results

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

Discussion

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.......

Materials

List of materials used in this article

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
27 G needle	Henke Sass Wolf	4710004020
1 mL syringe	Henke Sass Wolf	4010.200V0
Omnican 100 30 G 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