Name: establishment and optimization of a high throughput setup to study staphylococcus epidermidis and mycobacterium marinum infection as a model for drug discovery
Uploaded: 2014-06-26T14:00:00
Duration: 10 min 19 s
Description: Watch this Scientific Journal Video about Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discover

We are grateful to Leonie de Boer and Bas Zaat (Academic Medical Centre) for providing us with the S. epidermidis O-47 strain. We thank Rico Bongaarts, Francis Smet and Angela Comas (Union Biometrica) for help and advice with the COPAS XL and VAST BioImager analysis. We thank Davy de Witt, Ulrike Nehrdich, and Laura van Hulst for fish caretaking, and other colleagues from Leiden University for helpful discussions. This research forms part of the Project P5.03 IBIZA of the research program of the BioMedical Materials institute, co-funded by the Dutch Ministry of Economic Affairs, and of the Smart Mix Program (NWOA_6QY9BM) of The Netherlands Ministry of Economic Affairs and of The Netherlands Ministry of Education, Culture and Science. Additional support was obtained from the EU project ZF-Health (FP7-Health-2009-242048), and RMJ was supported by Marie Curie fellowship as Experienced Researcher in the EU Initial Training Network FishForPharma (PITN-GA-2011-289209). SJR received funding from the Innovative Medicines Initiative Joint Undertaking under grant agreement n&#176;115337, resources of which are composed of financial contribution from the European Union&#8217;s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind contribution.The authors further acknowledge financial support from the Leiden University Fund (LUF) for robotics and from Cyttron, in the Besluit Subsidies Investeringen Kennisinfrastructuur program, which in turn is financially supported by the Netherlands Organization for Scientific Research for imaging facilities. The COPAS system acquisition was partially supported by the Division for Earth and Life Sciences (ALW) with financial aid from the Netherlands Organization for Scientific Research (NWO, 834.10.004).

1. Bacterial Strains and Growth Conditions
<ol>
	<li>Prepare S. epidermidis&#160;Inoculum
	<ol>
		<li>Take several individual colonies from S. epidermidis strain O-47, containing a pWVW189 derived mCherry expression vector (De Boer L. unpublished) from a Luria Bertani (LB) agar plate supplemented with 10 &#956;g/ml chloramphenicol and culture overnight at 37 &#176;C in 25 ml LB medium supplemented with 10 &#956;g/ml chloramphenicol to midlog stage.</li>
		<li>Centrifuge 1 ml of the culture at 12,000 x&#160;g for 1 min and subsequently wash them 3x with 1 ml sterile phosphate buffered saline (PBS) with 0.3% (v/v) Tween-80.</li>
		<li>Measure the optical density at 600 nm (OD600), and dilute the bacterial suspension to an OD600 of 0.3 in 2% (w/v) polyvinylpyrrolidone40 (pvp40) in PBS. Note: An OD600 of 0.3 corresponds to 1.0 x&#160;108 colony forming units/ml (cfu/ml).</li>
	</ol>
	</li>
	<li>Prepare M. marinum&#160;Inoculum
	<ol>
		<li>Take several individual colonies from M. marinum strain M or E11 containing the pSMT3-mCherry vector stably expressing mCherry10 from a Middlebrook 7H10 agar plate with 10% (v/v) Middlebrook OADC enrichment supplemented with 50 &#956;g/ml hygromycin and culture overnight at 28 &#176;C in 10 ml of Middlebrook 7H9 broth with 10% (v/v) Middlebrook ADC enrichment supplemented with 50 &#956;g/ml hygromycin.</li>
		<li>Centrifuge 1 ml of the culture at 12,000 x&#160;g for 1 min and subsequently wash it 3x with 1 ml sterile PBS with 0.3% (v/v) Tween-80.</li>
		<li>Measure the OD600, and dilute the bacterial suspension to an OD600 of 0.3 in 2% (w/v) pvp40 in PBS. Note: An OD600 of 1 corresponds to 1.0 x&#160;108 cfu/ml.</li>
	</ol>
	</li>
</ol>
2. Prepare Zebrafish Eggs
<ol>
	<li>Place a maximum of 70 male and 50 female wild type zebrafish separately into the large breeding vessel. Note: Place the female fish in the lower part of the large breeding vessel.</li>
	<li>Remove the separator the next day in the morning, to let the zebrafish start breeding.</li>
	<li>Collect the eggs at the bottom of the large breeding vessel through the egg collector in a 50 ml tube filled with egg water (60 &#181;g/ml instant ocean sea salt).</li>
</ol>
3. Injection Needles
<ol>
	<li>Obtain commercially available custom made glass capillaries needles with an inner diameter of 10 &#181;m.</li>
</ol>
4. Experimental Outline of Injection
<ol>
	<li>Boil 100 ml of 1% (w/v) agarose in egg water, and cool until approximately 40 &#186;C. Pour agarose into the automated microinjectors plate and place the 1,024-well stamp into the agarose. Note: The plate is ready to use when cooled down.</li>
	<li>On the automated microinjector operating software click on &#8216;Calibrate stage&#8217;, then click on &#8216;1024&#8217; well grid and place the agarose plate in the microinjector and calibrate the plate by clicking on the screen at the center&#160;position of the well.</li>
	<li>Go to &#8216;needle menu&#8217; and click on &#8216;calibrate needle holder&#8217;.</li>
	<li>Fill the injection needle using a microloader tip with either 10 &#181;l pvp40 containing 100 cfu/nl S. epidermidis or 30 cfu/nl M. marinum, or use pvp40 as mock injection.</li>
	<li>Place the needle in the automated micro-injector and calibrate the x, y position by lowering or moving the needle up and clicking on the screen at the position of the needle. Then calibrate the z position of the needle by clicking on the screen at the position of the tip of the needle.</li>
	<li>Distribute the eggs over the agarose grid using a plastic transfer pipet, and remove excess egg water. Place the agarose grid in the automated microinjector.</li>
	<li>Go to the &#8216;Injection menu&#8217; and adjust the &#8216;Injection pressure&#8217; setting to 200 hPa, &#8216;Injection time&#8217; 0.2 sec and &#8216;Compensation pressure&#8217; 15 hPa, which correlates with 1 nl, at the Femtojet settings menu.</li>
	<li>Click on &#8216;Inject all&#8217; to inject the entire plate.</li>
	<li>Collect the eggs after injection by washing them into a Petri dish (92 x&#160;16 mm), with a maximum of 70 embryos per Petri dish, and incubate at 28 &#176;C.</li>
</ol>
5. Flow-cytometer Analysis
<ol>
	<li>Prepare the large particle flow cytometer according to the manufacturer&#8217;s instructions and fill the sample cup and sheath fluid container with egg water.</li>
	<li>At the operating software, go to the &#8216;PMT&#8217; menu and use the following settings: 650 V for the &#8216;Red&#8217; channel and 0 V for the &#8216;Green&#8217; and &#8216;Yellow&#8217; channels. Then go to &#8216;Thresholds&#8217; menu and use the following settings: &#8216;Optical density&#8217; threshold signal: 975 mV (COPAS XL value: 50) and the &#8216;Time Of Flight&#8217; (TOF) minimum to 320 &#956;sec (COPAS XL value: 800) in order to reduce the influence of debris.</li>
	<li>For analysis without sorting the embryos go to step 5.4, for analysis and sorting the embryos into a Petri dish go to step 5.5 or for analysis and sorting the embryos into a 96-well plate go to step 5.6.</li>
	<li>Place the embryos in the sample cup and click on &#8216;start&#8217; to start the analysis. When all embryos are analyzed stop the analysis by clicking on &#8216;stop&#8217;. Save the data by clicking on &#8216;Store all&#8217;. Note: All data are stored as TXT, LMD, DAT, CSV and BSRT files. Follow the protocol at step 5.7.</li>
	<li>Place the embryos in the sample cup and define the maximum of 70 embryos per plate to be sorted by entering 70 in the &#8216;Sort&#8217; menu. Place an empty Petri dish under the sorter and click on &#8216;Manual Sort&#8217;. When the Petri dish is filled, save the data by clicking on &#8216;Store all&#8217;. Note: All data is stored as TXT, LMD, DAT, CSV and BSRT files. Follow the protocol at step 5.7.</li>
	<li>Place the embryos in the sample cup and define the maximum of 1 embryo per well to be sorted by entering 1 in the &#8216;Sort&#8217; menu. Place an empty 96-well plate into the left plate holder and click on &#8216;Fill plate&#8217;. When the 96-well plate is filled, save the data by clicking on &#8216;Store all&#8217;. Note: All data is stored as TXT, LMD, DAT, CSV and BSRT files. Follow the protocol at step 5.7.</li>
	<li>Get the TXT file to process the raw data, use the following data filter settings: &#8216;Status select&#8217;: 40, and if using the sort module &#8216;Status sort&#8217;: 6. Then use the numbers from the total fluorescence signal from the &#8216;Red&#8217; channel to calculate the average and the standard error of the mean. Plot these data sets into bar or scatter graphs.</li>
</ol>
6. Drug Treatment
<ol>
	<li>Analyze and sort at 3 days post injection (dpi), M. marinum infected embryos in two equal groups using the large particle flow cytometer (step 5.5). Treat one group with a compound of interest in its carrier solvent and other with carrier solvent alone (control). Apply similar treatments to mock injected control to test for antibiotic side effects.</li>
	<li>Repeat at 4 and 5 dpi the analysis (step 5.5) and refresh the egg water or egg water containing the compound.</li>
</ol>
7. High Resolution Imaging
<ol>
	<li>Anesthetize the embryos with 0.02% (w/v) buffered 3-aminobenzoic acid ethyl ester (Tricaine) in egg water 10 min before analysis.</li>
	<li>Prepare the Vertebrate Automated Screening Technology system and the Large Particle Sampler according to the manufacturer&#8217;s instructions.</li>
	<li>Select the reference images corresponding to the age of the embryos from the &#8216;Imaging &#8211; Object &#8211; Detection Setup&#8217; menu.</li>
	<li>Select the amount of pictures and orientation to be made by the Vertebrate Automated Screening Technology system from the &#8216;Imaging &#8211; Auto store images&#8217; menu.</li>
	<li>Place a 96-well plate filled with embryos (from step 5.6) into the left plate holder of the Large Particle Sampler, and click on &#8216;Run plate&#8217;.</li>
	<li>When an embryo is detected and correctly positioned; image the head and the tail separately with the CLSM using a 10X plain dry objective and stitch the images afterwards using image processing software.</li>
</ol>

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J.S is owner of Life Science Methods BV that produces instruments used in this article.

The high throughput methodology described in this paper provides a fast and cost effective pipeline to screen high number of fish embryos and larvae with different types of infections. Using the large breeding vessel instead of traditional single or family breeding tanks facilitated control of the spawning process and generation of larger number of synchronous eggs. With an improved version of the automated micro-injection system7, it is possible to inject up to 2,500 eggs almost all in the same cell stage within 1 hr. With these updates and improved software it is feasible to inject more eggs than previously was possible which can be used to perform large drug screens with bacterial proliferation as a read out. However this method is still limited to yolk injection, other injection routes for example described by Benard et al. (2012)6, will hopefully be incorporated in the automated micro-injection system in the near future.
Although these methods are benchmarked for screening zebrafish, it would be useful for applications with other fish species as well. For instance, the common carp has been indicated to have advantages for drug screens. Like zebrafish, eggs and early stage embryos from common carp are transparent but with the main advantage of its large spawn size of hundred thousands of eggs and the availability of inbred lines that offer a more constant genetic background16.
The analysis of large amounts of infected embryos is done with the high throughput large particle flow cytometer. This device can sort analyzed embryos into multi well plates or a Petri dish making it especially suitable for testing large numbers of compounds. If a higher imaging resolution is needed, than the setup is adapted in a way that the large particle flow cytometer technology can be used for pre-screening and subsequently analyze the samples at a medium throughput at a higher resolution. This can be done using the Vertebrate Automated Screening Technology17,18. This device can automatically collect live or fixed embryos between 2 and 7 days post fertilization from a multi well plate or bulk container, image 360&#176; through a capillary using CLSM or stereo microscopy and dispose again in 2 bulk containers allowing manual sorting of the embryos based on the microscopic images. Future improvements will allow the sorting of the embryo after imaging into the multi well plate, therefore making it possible to screen automatically large number of individual embryos over time with CLSM. Assuming that in future applications the Vertebrate Automated Screening Technology system can also be connected to the large particle flow cytometer technology without the need of prior dispensing larvae into multi well plates, will lead to a more advanced sorting.
This paper describes the establishment and optimization of a high throughput setup to study S. epidermidis and M. marinum infection as a model for drug discovery. It demonstrates that the outcome of these bacteria injected into the yolk depends on the developmental stage of the eggs at the time of injection. Injecting M. marinum E11 at 16-128 cell stage or the M strain at 16-64 cell stage leads to the same infection pattern as caudal vein injection2,6. However this setup is not limited to the proliferation of bacterial pathogens only. It was shown before that it is possible to robotically inject solutions containing DNA, RNA or morpholinos for transgenesis, over-expression and gene knock-down studies, respectively13. Furthermore, it was shown that this setup is also useful for the study of cancer cell proliferation and migration. Therefore this pipeline presents a versatile method for high throughput screens of a variety of signal mechanisms in the context of innate immunity, applied to infectious disease and the development of cancer. These screens can be combined with others for medicine discovery but also with analysis of possible toxic effects of identified applicable drugs.

To date the zebrafish (Danio rerio) has been successfully established as an efficient model to study a variety of infectious diseases1. The zebrafish embryo offers unique in vivo imaging possibilities due to their transparency and the large number of existing transgenic reporter lines expressing fluorescent proteins. This powerful combination makes it possible to track different immune cell types in time while interacting with pathogens such as Mycobacterium marinum, the closest relative of M. tuberculosis2, or Staphylococcus epidermidis, the main causative of biomaterial associated infection3-5. Different routes of infection can be used in zebrafish embryos depending on the purposes of the study6.
One of these infection routes is yolk injection of the bacteria. The main advantage of this method compared to the others is that yolk infection can be performed automatically via robotic injection, significantly reducing the injection time and allowing high reproducibility of the infection7,8.
Previous work, using the zebrafish as a high throughput in vivo model system for the study of S. epidermidis and M. marinum infection showed to be successful7,8. This system is able to screen for disease progression via robotic yolk injection of early embryos and using fluorescence readout as a measure for the bacterial load. In agreement with this notion, this setup has been optimized and established a highly efficient high throughput pipeline with the potential to generate large numbers of homogenously infected embryos and track the progression of the infection during the time after treatment with a number of compounds. With the established setup it is possible to generate up to 8,000 synchronous embryos to screen for disease progression, processing in this way up to 2,500 embryos per hour. Embryos are sorted based on their bacterial load using an automated system, ensuring homogenous groups of infected larvae. Furthermore, to validate the setup, effects of reference known to prevent tuberculosis progression in mammals have been tested on embryos infected with M. marinum E11 strain or the more virulent M strain9.
This study describes in detail the high throughput pipeline that has been established to be able to generate large numbers of infected embryos and the subsequent analysis of the bacterial progression during development and after compound treatment.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Middlebrook 7H10 agar</td>
			<td>BD Bioscience, Franklin Lakes, New Jersey, USA</td>
			<td>262710</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 broth</td>
			<td>BD Bioscience, Franklin Lakes, New Jersey, USA</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Middlebrook oleic acid albumin dextrose catalase (OADC) enrichment</td>
			<td>BD Bioscience, Franklin Lakes, New Jersey, USA</td>
			<td>211886</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Middlebrook albumin dextrose catalase (ADC) enrichment</td>
			<td>BD Bioscience, Franklin Lakes, New Jersey, USA</td>
			<td>211887</td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar</td>
			<td>Sigma-Aldrich, Saint Louis, Missouri, USA</td>
			<td>L5542</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Sigma-Aldrich, Saint Louis, Missouri, USA</td>
			<td>L3022</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Bio-connect, Huissen, the Netherlands</td>
			<td>16785.03</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr>
			<td>Hygromycin</td>
			<td>Bio-connect, Huissen, the Netherlands</td>
			<td>25966.01</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Sigma-Aldrich, Saint Louis, Missouri, USA</td>
			<td>P1754</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone40</td>
			<td>Calbiochem, San Diego, California, USA&#160;</td>
			<td>529504</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td>Sigma-Aldrich, Saint Louis, Missouri, USA</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sphaero-Q, Gorinchem, the Netherlands</td>
			<td>S103</td>
			<td>Multiple suppliers</td>
		</tr>
		<tr>
			<td>Instant ocean sea salt</td>
			<td>Sera Marin, Heinsberg, Germany</td>
			<td>5460</td>
			<td></td>
		</tr>
		<tr>
			<td>iSPAWN</td>
			<td>Techniplast, Buguggiate, Italy</td>
			<td>iSPAWN</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated microinjection system</td>
			<td>Life Science Methods BV, Leiden, the Netherlands</td>
			<td>Automated microinjection system</td>
			<td></td>
		</tr>
		<tr>
			<td>Complex Object Particle Analyzer and Sorter XL (COPAS XL)</td>
			<td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td>
			<td>COPAS XL</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertebrate Automated Screening Technology BioImager (VAST BioImager)</td>
			<td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td>
			<td>VAST BioImager</td>
			<td></td>
		</tr>
		<tr>
			<td>LP Sampler</td>
			<td>Union BioMetrica Inc., Holliston, Massachusetts, USA</td>
			<td>LP Sampler</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>TCS SL</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle 10 &#181;m inner diameter</td>
			<td>Qvotek, Mississauga, Canada</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment and optimization of a high throughput setup to study staphylococcus epidermidis and mycobacterium marinum infection as a model for drug discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

The present results show that the high throughput pipeline to study S. epidermidis and M. marinum infection has been successfully established and that may be extended to other infection models. Firstly, the use of the large breeding vessel (Figure 1A), based on the published system by Adatto et al. (2011)11, enables to generate large numbers of synchronous eggs in single events affording a high control of the spawning process. Next to be able to inject large number of embryos in a short period of time, an improved version of the previously developed automated micro-injection system7 was used (Figure 1A). To asses which is the best developmental stage for yolk infection, injections with S. epidermidis and M. marinum were performed at all the different stages between 1 and 512 cell stage, according to the description made by Kimmel et al. (1995)12.
Injections with 100 cfu S. epidermidis between the 16 and 128 cell stage provided the best infection pattern (Figure 2). The bacteria proliferated inside the yolk for 3 days and spread into the body from 3 dpi onwards. Performing injections before the 16 cell stage led to high mortality from 4 dpi, and injection after the 256 cell stage showed mainly bacterial growth inside the yolk with hardly any bacteria spreading inside the body of the embryo. Quantification of bacterial burden was performed by fluorescence intensity analysis using the large particle flow cytometer as described by Veneman et al. (2013)8 (Figure 3).
Observations showed that the optimal developmental stage for injection of 30 cfu M. marinum injection is between 16 to 128 cell stage for the E11 strain (Figure 4A) and between 16-64 cell stage with the more virulent M strain (Figure 4B). Embryos injected at these stages showed bacterial growth inside the yolk and spreading of the bacteria through the embryo (Figure 7). Infection with both strains at earlier stages presented nonspecific generalized bacterial growth leading the embryos to die after 4 dpi. On the other hand, in embryos injected at later stages bacterial burden was restricted to the yolk.
Next, presorting with large particle flow cytometer (Figure 1B) generated large homogenous groups of infected fish excluding non- or highly infected embryos (Figures 5A and 6A). After presorting, M. marinum infected embryos were treated with Rifampicin, a first line antituberculosis drug. Previous studies demonstrated that treatment with Rifampicin at a dose of 200 &#956;M efficiently reduces M. marinum infection in zebrafish7,13. Taking advantage of the large number of homogenously infected embryos generated with the high throughput setup, treatment with different doses was performed. Embryos infected with M. marinum M strain and treated for 48 hr&#160;with 12, 24, and 200 &#956;M Rifampicin showed to reduce efficiently mycobacterial infection in a dose dependent manner (Figure 5B). In view of the efficient reduction of the infection using Rifampicin at a dose of 200 &#956;M this concentration was used for the future experiments. In line with the previous result, studying bacterial burden progression using M. marinum E11 strain a significant reduction 24 hr&#160;and onwards after treatment with 200 &#956;M Rifampicin was observed (Figure 6B). &#160;
Furthermore, if high magnification imaging is required of these embryos, they can be displayed automatically in 96-well plates (Figure 1C), from where the samples can be analyzed using the Vertebrate Automated Screening Technology system with the Large Particle Sampler mounted onto a CLSM.
The Vertebrate Automated Screening Technology system with the Large Particle Sampler is a system that can either be mounted onto a CLSM or stereo microscope. This device allows the loading of live or fixed embryos from a 96-well plate or bulk container automatically through a glass capillary, and orientates it in front of the camera at the desired angle (e.g., dorsal or lateral). Images of the embryo in all orientations can be made with the on board camera or with an external CLSM (Figure 7). Embryos will subsequently be transferred in the collection or waste container.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig1highres.jpg" src="/files/ftp_upload/51649/51649fig1.jpg" /> 
Figure 1. Mainstream experimental outline. A) Adult fish are put together to mate, eggs are collected, aligned into an agarose plate and injected. B) The injected eggs are incubated at 28 &#176;C and will be pre-sorted for possible drug treatment. C) Subsequent analysis by large particle flow cytometer and/or Large Particle Sampler/Vertebrate Automated Screening Technology with CLSM. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig2highres.jpg" src="/files/ftp_upload/51649/51649fig2.jpg" /> 
Figure 2. Establishment of best cell stage for S. epidermidis yolk injection. Zebrafish embryos were injected in the yolk at different developmental stages from 1 to 512 cell stage with 100 cfu of S. epidermidis. Embryos injected between 1 and 8 cell stage showed bacterial growth in the yolk and high mortality from 4 dpi. Embryos injected between 16 and 128 cell stage showed bacterial growth in the yolk and inside the body starting at 3 dpi. Embryos injected between 256 and 512 cell stage showed many bacterial growth inside the yolk. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig3highres.jpg" src="/files/ftp_upload/51649/51649fig3.jpg" /> 
Figure 3. Quantification of bacterial burden using large particle flow cytometer. 100 cfu of S. epidermidis were injected into the yolk of zebrafish embryos. A) Up to 5 dpi, each day, groups of 10 embryos were homogenized and plated directly, showing the average exponential growth based on two biological replicas (error bars = SEM). B) Large particle flow cytometer analysis shows the average fluorescence signal from non-injected and S. epidermidis injected embryos. 30-160 embryos per condition were analyzed (error bars = SEM), different letters indicate statistical significant differences by one way ANOVA followed by Tukey&#39;s post-hoc test (P &#60; 0.001), ns: not significant differences. C) Correlation between cfu and average fluorescence signal of groups of 10 S. epidermidis infected embryos (error bars = SEM). This figure has been modified from Veneman et al. (2013)8. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig4highres.jpg" src="/files/ftp_upload/51649/51649fig4.jpg" /> 
Figure 4. Establishment of the best cell stage for M. marinum yolk injection. Zebrafish embryos were injected at all the different developmental stages from 1 to 512 cell stage with 30 cfu of M. marinum E11 and M strain. A, B) Embryos injected from 1-8 cell stage showed similar spreading and mortality with both strains. A) Embryos injected between 16-128 cell stage with E11 strain showed formation of granulomas and systemic infection while those injected from 256 to 512 cell stage kept bacterial burden into the yolk. B) Embryos injected between 16-64 cell stage with M strain showed formation of granuloma like structures and systemic infection while those injected from 128 to 512 cell stage kept bacterial burden into the yolk. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig5highres.jpg" src="/files/ftp_upload/51649/51649fig5.jpg" /> 
Figure 5. Treatment of M. marinum acute infection with a first-line anti-tuberculosis drug. Embryos injected between 16-64 cell stage with 30 cfu of M. marinum M strain were run through the large particle flow cytometer at 3 dpi to be sorted in two groups after discarding the non- and/or highly infected embryos. A) Fluorescence of individual embryos in both groups. B) Embryos treated with Rifampicin (RIF) for 48 hr at doses of 12, 24, and 200 &#956;M were analyzed at 4 dpi; their bacterial load is significantly reduced. C) Representative COPAS profiles of embryos treated with DMSO and Rifampicin at doses of 12, 24, and 200 &#956;M for 24 hr. Bacterial load and distribution is indicated by the red peaks. Blue line represents the profile of the element sorted (4 dpf zebrafish embryo) by the COPAS. 60-90 embryos per condition were analyzed. Each data point represents an individual embryo. Values are indicated as mean &#177; SEM. ns: not significant differences. Analysis of statistical significance of differences was performed by one way ANOVA followed by Tukey&#39;s posthoc test. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig6highres.jpg" src="/files/ftp_upload/51649/51649fig6.jpg" /> 
Figure 6. Treatment of M. marinum chronic infection with a first line antituberculosis drug. Embryos injected between 16-64 cell stage with 30 cfu of M. marinum E11 strain were run through the large particle flow cytometer at 3 dpi to be sorted in two groups after discarding the non- and/or highly infected embryos. A) Fluorescence of individual embryos in both groups. B) Embryos treated with Rifampicin (RIF) at 200 &#956;M during 4 days were analyzed showing a significant reduction of the bacterial load after 1 day of treatment. 90 embryos per condition were analyzed. Values are indicated as mean &#177; SEM. Different letters indicate significant differences between time points of the same treatment. * indicates significant differences with control group. ns: not significant differences. Analysis of statistical significance of differences was performed by one way ANOVA followed by Tukey&#39;s posthoc test. (P &#60; 0.05). Figure B) has been modified from Spaink et al. (2013)13. <a href="https://www.jove.com/files/ftp_upload/51649/51649fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" fo:content-width="5in" fo:src="/files/ftp_upload/51649/51649fig7highres.jpg" src="/files/ftp_upload/51649/51649fig7.jpg" /> 
Figure 7. Result of M. marinum E11 yolk injection imaged using Vertebrate Automated Screening Technology and CLSM. Confocal Z stack (stitched 3 images) of a 5 dpi fli1-egfp14 embryo. A) Live embryo showing proliferation of M. marinum E11 bacteria (red) throughout the body. B) Fixed 5 dpi fli-egfp embryo showing M. marinum E11 bacteria (red) throughout the body co-localizing with leucocytes (light blue) detected by L-plastin immunostaining15.

Watch this Scientific Journal Video about Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discover

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

establishment-optimization-high-throughput-setup-to-study

Research

JoVE Journal

Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

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 ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

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.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

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.

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.

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