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 Sa&#776;a&#776;tio&#776;) (H.L., M.M.H., M.P.), Sigrid Juse&#769;lius Foundation (M.P.), Emil Aaltonen Foundation (M.M.H.), Jane and Aatos Erkko Foundation (M.P.) and Academy of Finland (M.P.). Leena Ma&#776;kinen, Hanna-Leena Piippo and Jenna Iloma&#776;ki are acknowledged for their technical assistance.&#160;The authors acknowledge the Tampere Zebrafish Laboratory for their service.

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.
<ol>
	<li>Culture M. marinum on a 7H10 plate supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment and 0.5% v/v of glycerol at 29 &#176;C for at least 5 days. Maintain M. marinum cultures by taking fresh bacteria from the freezer every two weeks and transferring onto a new plate every other week. 
	NOTE: M. marinum is a natural fish pathogen infecting aquatic species and it is important to take precautions not to contaminate zebrafish stocks with the bacteria. Infected zebrafish and items contaminated with bacteria have to be kept separate from the breeding facilities.</li>
	<li>Use a sterile 1-&#181;L inoculation loop to aseptically transfer a loopful of M. marinum bacterial mass into a cell culture flask containing 10 mL of 7H9 medium with 10% ADC (albumin, dextrose, catalase) enrichment, 0.2% v/v polysorbate 80 and 0.2% v/v of glycerol. Culture for 3&#8211;4 days to approximately an OD600 (optical density) of 0.7 at 29 &#176;C in the dark without shaking. Leave the cap loose, or use a filter cap, to allow for the sufficient exchange of gases. 
	NOTE: Polysorbate 80 is added to the medium to prevent the aggregation of bacteria. In addition, exposure to light leads to phenotypic changes in bacterial colonies (e.g., the color changes from white to yellow). To avoid this, keep the cultures in the dark.</li>
	<li>Measure the OD600value with a spectrophotometer. Dilute the liquid culture to an OD600 of 0.07&#8211;0.09 and continue culturing for 2 days at 29 &#176;C in the dark without shaking. Leave the cap loose. 
	NOTE: During these two days, the bacterial suspension will reach an OD600of approximately 0.5 corresponding to an early log-phase.</li>
</ol>
2. Preparation of Bacterial Solution for Infecting Adult Zebrafish
<ol>
	<li>Transfer the bacterial suspension into a big sterile cuvette or a 15 mL tube and place it in the dark at room temperature for 15 min to allow the biggest clumps to settle.</li>
	<li>Transfer the top 5&#8211;7 mL of the suspension into a clean tube or a cuvette and measure the OD600. Use this top phase of the suspension for the infections.</li>
	<li>Collect 1 mL of M. marinum culture into a fresh tube and centrifuge for 3 min at 10,000 x g. Remove the supernatant and resuspend the pellet in 1 mL of sterile 1x PBS.</li>
	<li>Dilute to reach the desired bacterial concentration by using sterile 1x PBS with 0.3 mg/mL phenol red as a tracer. Divide the diluted suspension into three aliquots. 
	NOTE: Use a predetermined OD600 vs. CFU (colony-forming units)/&#181;L curve to estimate the dilution required to get the wanted number of bacteria in the 5 &#181;L injection volume34. The correlation between OD600 and concentration of the bacterial suspension needs to be validated before starting the actual infection experiments. Reserve two weeks for collecting this validation data.</li>
	<li>Using a 1 mL syringe, slowly pull the suspension through a 27 G needle 3x. For each aliquot, perform this step just before use. 
	NOTE: Do not use the same bacterial solution for more than 2 h.</li>
</ol>
3. Experimental M. marinum Infection with Intraperitoneal Injection
<ol>
	<li>Pipette a 5 &#181;L droplet of the diluted bacterial solution onto a piece of parafilm film and pull the droplet into a 30 G insulin needle.</li>
	<li>Use 5&#8211;8 month-old wild-type fish and rag1&#8722;/&#8722;hu1999 mutant fish for the experiment. Anesthetize adult zebrafish in the tank water with 0.02% 3-aminobenzoic acid ethyl ester (pH 7.0). Position the fish ventral side up into a slit on a moist foamed plastic. 
	NOTE: Rag-/- mutant fish are not able to undergo somatic recombination and produce functional T and B cells.</li>
	<li>Inject the needle between the pelvic fins at an approximately 45&#176; angle. Keep the needle opening upwards to observe that the entire opening is inside the abdominal cavity. Slowly inject the bacterial suspension and carefully remove the needle. 
	NOTE: In case the red tracer is leaking out of the fish upon injection, exclude the fish from the experiment.</li>
	<li>Immediately after injection, transfer the fish into a recovery tank with fresh tank water.</li>
	<li>Take samples of the bacterial suspension on 7H10 plates every 15 min from the bacterial aliquot in use and incubate the bacteria at 29 &#176;C for 5 days and verify the infection dose by counting the colonies on the plates.</li>
	<li>Check the well-being of the fish regularly and euthanize any fish with symptoms of the infection with over 0.02% concentration of 3-aminobenzoic acid ethyl ester (pH 7.0). 
	NOTE: Approximately, 7% of the adult zebrafish infected with 34 &#177; 15 CFU and 30% of zebrafish infected with 2029 &#177; 709 CFU will have had symptoms by 8 weeks19. The symptoms may include abnormal swimming, lack of response to touch, gasping, edema or observable wasting.</li>
	<li>Maintain the zebrafish according to the common standards35.</li>
</ol>
4. Collection of Internal Organs
<ol>
	<li>Euthanize the zebrafish with an overdose of 3-aminobenzoic acid ethyl ester (over 0.02% concentration, pH 7.0) in the tank water.</li>
	<li>Insert one pin posterior to the branchiostegal rays and another through the tail to tack the fish onto the platform.</li>
	<li>Open the whole abdominal cavity with a scalpel and collect the internal organs by using a small spoon and sharp-ended tweezers. Start from the heart and work along the spine towards the tail to detach all internal organs in one block. 
	NOTE: Be sure to collect all kidney tissue by scraping along the spine with the spoon.</li>
	<li>Finally, use tweezers to detach the gut next to the cloaca and transfer the organs into a 1.5 mL homogenization tube with six 2.8 mm ceramic beads. Immediately place on dry-ice to freeze the sample. The sample can be stored at -80 &#176;C until homogenized.</li>
	<li>Rinse the instruments with 70% ethanol between individuals.</li>
</ol>
5. Homogenization and RNA extraction from an organ block.
NOTE: The method is modified from the Stanford University protocols36.
<ol>
	<li>Add guanidine thiocyanate-phenol solution used for nucleic acid extraction (Table of Materials) on the top of the sample to a total volume of 1,500 &#181;L. Ensure that the sample covers a maximum of 10% of the total volume. 
	CAUTION: The expected volume of the harvested tissue is 100 &#181;L. Guanidine thiocyanate-phenol solution contains toxic and irritating compounds and requires protective clothing, nitrile gloves and working in a fume hood. Do not combine with bleach as this will cause formation of toxic gases. Read the material safety data sheet (MSDS) before use.</li>
	<li>Homogenize samples using a bead-beating homogenizer 3 times for 40 s at 3,200 rpm. Cool on ice for 30 s between the cycles. Sonicate the homogenized samples in a water bath for 9 min.</li>
	<li>Centrifuge the samples at 12,000 x g for 10 min at 4 &#176;C and move 1,000 &#181;L of the cleared homogenate into a fresh microcentrifuge tube.</li>
	<li>Add 200 &#181;L of chloroform, immediately mix by vortexing for 15 s and incubate for 2 min at room temperature. 
	CAUTION: Chloroform is a toxic and irritant compound if inhaled, swallowed or contacted with skin or eyes. Use necessary safety equipment for personal protection and work in a fume hood. Read the material safety data sheet (MSDS) before use.</li>
	<li>Centrifuge at 12,000 x g for 15 min at 4 &#176;C to separate the aqueous and organic phases.</li>
	<li>Carefully, transfer 500 &#181;L of the top phase to a fresh tube to avoid contaminating the RNA. Remove and discard the rest of the aqueous phase (~100 &#181;L) and store the interphase and organic phase at 4 &#176;C for DNA extraction. 
	NOTE: The top phase contains the RNA.</li>
	<li>Add 500 &#181;L of 2-propanol and immediately mix by vortexing for 15 s. Incubate for 10 min at room temperature to precipitate the RNA.</li>
	<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C to pellet the RNA. Remove the supernatant by pipetting.</li>
	<li>Add 1 mL of 75% ethanol and vortex for 10 s. 
	NOTE: The protocol can be paused here, and the samples kept overnight at 4 &#176;C.</li>
	<li>Centrifuge at 7,500 x g for 5 min at 4 &#176;C. Remove the supernatant by pipetting.</li>
	<li>Repeat the wash steps 5.9&#8211;5.10. Remove the supernatant carefully by pipetting and let the pellet air-dry in a fume hood.</li>
	<li>Dissolve the RNA pellet in 500 &#181;L of nuclease-free water and keep the samples on ice. Measure the concentrations with a microvolume spectrophotometer or with equivalent equipment. Store the RNA at -80 &#176;C.</li>
</ol>
6. Purification of Co-extracted Zebrafish and Mycobacterial DNA
<ol>
	<li>Prepare a back-extraction buffer (BEB) by dissolving 118.2 g of guanidine thiocyanate (final concentration 4 M), 3.68 g of sodium citrate (final concentration 50 mM) and 30.29 g of Tris free base (final concentration 1 M) in 120 mL of nuclease-free water (this may require stirring overnight). Add nuclease-free water to a final total volume of 250 mL and filter to sterilize the solution. 
	NOTE: This buffer can be stored at room temperature for up to 6 months. Do not combine BEB with bleach as they react to produce toxic gases such as hydrogen chloride and hydrogen cyanide.</li>
	<li>Use the interphase and organic phase of the sample to extract mycobacterial DNA. Add 500 &#956;L of BEB to each tube. Mix extensively for 10 min by inversion at room temperature.</li>
	<li>Centrifuge the tubes at 12,000 x g for 30 min at room temperature and carefully transfer 500 &#181;L of the upper aqueous phase containing the DNA to a new tube.</li>
	<li>Add 400 &#956;L of 2-propanol. Mix by inverting and incubate for 10 min at room temperature.</li>
	<li>Centrifuge the samples at 12,000 x g for 15 min at 4 &#176;C. A pellet containing the DNA should be visible at this point. Carefully remove the supernatant by pipetting.</li>
	<li>Add 800 &#956;L of 70% ethanol. Wash the pellet by inversion. Do not vortex the samples at this point, as genomic DNA breaks down easily.</li>
	<li>Centrifuge the samples at 12,000 x g for 15 min at 4&#176;C and remove the supernatant by pipetting. Repeat the ethanol wash (steps 6.6 and 6.7).</li>
	<li>Remove the ethanol by carefully pipetting. Let the samples air-dry for 5&#8211;10 min. Dissolve the pellet in 200 &#956;L of nuclease-free water.</li>
	<li>Measure DNA concentrations with a microvolume spectrophotometer or with equivalent equipment. DNA can be stored at 4 &#176;C or at -20 &#176;C for long-term storage.</li>
</ol>
7. Quantitative PCR for Measuring Mycobacterial Loads
<ol>
	<li>Prepare qPCR reaction mixes with no-ROX (carboxy-X-rhodamine) against the M. marinum internal transcribed spacer (ITS) between 16S-23S ITS according to the manufacturer&#39;s instructions with MMITS1 primers (Table 1). Pipette the reaction mix and sample dilutions as duplicates on a 96-well plate suitable for qPCR. Include a DNA standard dilution series of a known amount of bacteria in each run. 
	NOTE: The expected M. marinum load per fish can range from 0 CFU to 1,000,000 CFU at 4 wpi. The qPCR assay can be also performed with other qPCR kits but the annealing temperature for the primers has to be re-optimized.</li>
	<li>Seal the plate with an optically transparent film and centrifuge the plate at 2,000 x g for 2 min at 4 &#176;C.</li>
	<li>Run the qPCR program shown in the Table 2.</li>
	<li>Using the standard curve, calculate the number of bacteria in the entire fish sample.</li>
</ol>
8. DNase Treatment of the RNA Samples
<ol>
	<li>To remove any possible remaining traces of genomic DNA from the RNA, carry out DNase I treatment. Thaw the RNA samples on ice. 
	NOTE: Be sure to use only RNase-free equipment and solutions and wipe the working surface and pipettes with a decontamination reagent eliminating RNases (Table of Materials) before starting to work. Wear a long-sleeved lab coat and gloves to protect your samples.</li>
	<li>Prepare 10 &#956;L DNase I reaction mixes on ice according to the manufacturer&#39;s instructions. Mix 1 &#956;L of DNase I, 1 &#956;L of 10x DNase buffer and 8 &#956;L of RNA sample including a maximum of 1 &#956;g of RNA.</li>
	<li>Gently mix the reactions (no vortexing) and incubate for 30 min at 37 &#176;C.</li>
	<li>Prior to heat-inactivation, add 1 &#181;L of 50 mM EDTA to each 10 &#181;L sample. If EDTA is not added, the RNA will undergo chemical degradation when heated.</li>
	<li>Incubate for 10 min at 65 &#176;C to heat-inactivate DNase I. Continue directly to cDNA synthesis or store the DNase-treated RNA at -80 &#176;C.</li>
</ol>
9. cDNA Synthesis
<ol>
	<li>Keep all reagents and samples on ice and prepare the reaction mixes according to the manufacturer&#39;s instructions. For a 5 &#956;L reaction mix, include 1 &#956;L of Reverse Transcription Master Mix, 3 &#956;L of nuclease-free water and 1 &#956;L of DNase treated RNA.</li>
	<li>Gently mix the reverse transcription reactions and briefly spin the tube, if needed.</li>
	<li>Place samples in a PCR machine and use the program shown in Table 3.</li>
	<li>Dilute the cDNA in nuclease-free water for qPCR to a maximum concentration of 2.5 ng/&#956;L, if needed. cDNA can be stored at -20 &#176;C.</li>
</ol>
10. Measuring Zebrafish Gene Expression by Quantitative PCR
<ol>
	<li>Prepare a qPCR master mix on ice according to the manufacturer&#39;s instructions and protect from light. Use the primers introduced in Table 1. 
	NOTE: To calculate the fold of induction for each gene, measure the expression also from a pooled baseline sample extracted from 6 healthy zebrafish.</li>
	<li>Prepare replicates of each sample and pipette the reaction mixes onto a qPCR plate. Seal the plate with an optically transparent film and centrifuge the plate at 2,000 x g for 2 min at 4 &#176;C before starting the run.</li>
	<li>Run the qPCR program shown in the Table 4 with the annealing temperature depending on the primer pair used (Table 1).</li>
	<li>Analyze the gene expression ratio compared to a house-keeping gene (loopern437) with the &#916;Ct method using the equation: 
	<img alt="Equation" src="/files/ftp_upload/58299/58299eq1.jpg" /></li>
</ol>

The authors have nothing to disclose.

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...

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 toxicology7,8.
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 tuberculosis18,19,20,21,22. 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. Mice24,25, 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 organs19,30. 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 tuberculosis19,31,32. 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.

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

modeling tuberculosis in mycobacterium marinum infected adult zebrafish

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.

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 <strong class="xfig"...

Watch this Scientific Journal Video about Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish at JoVE.com

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

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&#39;s immune responses with qPCR.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

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Immunology and Infection

10.5K Views.  University of Tampere. 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.

Video: Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

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The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.