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 fo...

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 ...

This method can help to answer key questions in the field of tuberculosis such as what is the role of adaptive immune responses and the pathogenesis of this multifactorial disease. The main advantage of this nucleic acid base method is that both bacterial loads and gene expression levels can be measured from the same individual. To begin this procedure, culture M.marinum on a 7H10 plate as outlined in the text protocol.Transfer 10 milliliters of 7H9 medium containing ADC enrichment, polysorbate 80, and glycerol to a cell culture flask. Then, use a sterile one microliter inoculation loop to aseptically transfer one loopful of the M.marinum bacterial mass into the flask. Leave the cap loose or use a filter cap to allow for sufficient gas exchange and culture at 29 degrees Celsius in the dark without shaking for three to four days until the OD600 reaches about 0.7.Dilute and continue culturing the bacteria as outlined in the text. To begin preparing the bacterial solution for infection, transfer one milliliter of the culture into a fresh tube. Centrifuge at 10, 000 g for three minutes.Remove the supernatant and resuspend the pellet in one milliliter of sterile PBS. Using sterile PBS with a 0.3 milligrams per milliliter of phenol red as a tracer, dilute the suspension to reach the desired bacterial concentration. After this, use a one milliliter syringe to slowly pull the suspension through a 27 gauge needle three times.Perform this step just before use for each aliquot. First, pipette a five microliter droplet of the diluted bacterial solution onto a piece of paraffin film. Then, pull the droplet into a 30 gauge insulin needle.Use a five to eight-month-old fish for this experiment with one being a wild type fish and the other being a rag mutant fish. Position these fish ventral side up in the slits of a piece of moist foamed plastic. Inject the insulin needle between the pelvic fins at a 45 degree angle.Keep the needle opening upwards to ensure that the entire opening is inside the abdominal cavity. Then, slowly inject the bacterial solution. After this, carefully remove the needle and immediately transfer the fish to a recovery tank filled with fresh tank water.Take samples from the bacterial aliquot in use every 15 minutes on 7H10 plates. Incubate these samples at 29 degrees Celsius for five days to verify the infection dose. Check the wellbeing of the fish regularly, making sure to euthanize any fish with infection symptoms by incubating them in water with more than 0.02%of 3-aminobenzoic acid ethyl ester.After euthanizing the fish, insert a pin posterior to the branchiostegal rays. Insert a second pin through the tail to tack the fish onto the platform. Using a scalpel, open the whole abdominal cavity.Then, use a small spoon and sharp-ended tweezers to collect the internal organs starting at the heart and working along the spine towards the tail to detach all of the internal organs in one block. Use tweezers to detach the gut net from the cloaca and transfer the organs into a 1.5 milliliter homogenization tube containing half a dozen 2.8 millimeter ceramic beads. Immediately place the tube on dry ice to freeze the sample.Store the sample at negative 80 degrees Celsius until ready to homogenize. After extracting the DNA and RNA, measure the mycobacterial loads by quantitative PCR as outlined in the text protocol. In this study, adult zebrafish are infected with M.marinum by an intraperitoneal injection.A high infection dose is seen to lead to a progressive disease in which the mycobacterial load continues to increase until the average load reaches about five million bacteria ultimately killing the fish. A low dose on the other hand leads to the development of a disease spectrum similar to that seen in human tuberculosis with progressive, latent, reactivated, and sterilized infections. In this case, the load continues to increase for four weeks after which the disease reaches a steady state in the majority of the fish.This data reveals that the initial number of mycobacteria used to infect the fish is a critical determinant for the outcome of the infection. Rag mutant zebrafish are seen to be unable to sufficiently limit the growth of mycobacteria leading to higher bacterial loads and increased morbidity. This clearly demonstrates the importance of adaptive immunity in controlling mycobacterial infection.The levels of interleukin-4 are significantly higher in the wild type group revealing that the adaptive response is required for the efficient introduction of interleukin-4, but dispensable for the introduction of interferon gamma after mycobacterial infection. This protocol enables the collection of both DNA and RNA from the same sample which makes it possible to combine the mycobacterial burden of the sample with gene expression data of both the host and the bacteria. This technique can be combined with the administration of drugs or vaccine candidates to assess their effect on the mycobacterial loads and immune responses.While attempting this procedure, it is important to remember to follow the local guidelines for personal safety and waste disposal when working with a biosafety level two pathogen. This technique makes it possible to study both active and latent tuberculosis with dormant mycobacteria in the zebrafish which is an ethical non-mammalian in vivo model.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Abstract