This work was supported by grants from the Spanish Ministry of Science, European commission and AGAUR funds to NR (AGL2015-65129-R MINECO/FEDER and 2014SGR-345 AGAUR). RT holds a pre-doctoral scholarship from AGAUR (Spain), JJ was supported by a PhD fellowship from the China Scholarship Council (China) and NR is supported by the Ram&#243;n y Cajal program (RYC-2010-06210, 2010, MINECO). We thank Dr. Torrealba for expert advice in protein production, N. Barba from the "Servei de Microscopia" and Dr. M. Costa from the "Servei de Citometria" of the Universitat Aut&#242;noma de Barcelona for helpful technical assistance.

All experimental procedures involving zebrafish (Danio rerio) were authorized by the Ethics Committee of the Universitat Aut&#242;noma de Barcelona (CEEH number 1582) in agreement with the International Guiding Principles for Research Involving Animals (EU 2010/63). All experiments with live zebrafish were performed at 26&#8211;28 &#176;C.
1. Preparing the Equipment for Oral Intubation
<ol>
	<li>Place approximately 1 cm of a fine silicone tube on a 31 G Luer lock needle to cover the needle tip.</li>
	<li>Cut a 10 &#181;L sterile filter pipette tip (around 2 cm), take the finer end and place it over the silicone tube as a sheath. Make sure the pipette extends beyond the tip of the needle to avoid injuring the animal.</li>
	<li>Attach the needle to a 100 &#181;L Luer lock syringe. 
	NOTE: Always rinse with ethanol and then phosphate buffered saline (PBS, see Materials) thoroughly between treatments.</li>
</ol>
2. Solutions Required
<ol>
	<li>Prepare 150 mg/L (for anesthesia) or 300 mg/L (for euthanasia) of ethyl 3-aminobenzoate methanesulfonate (MS-222) solution with water from the aquarium where the zebrafish are maintained. Fill a small tank with 1 L of anesthetic solution and keep it aerated.</li>
	<li>Fill another small tank with 1 L of aquarium water without MS-222 for fish recovery and keep it aerated.</li>
	<li>Make 50 mL of 1x PBS from a 10x sterile stock solution.</li>
	<li>For cytometry analysis/intestinal cell isolation, prepare enough fresh 0.15% collagenase Type IV solution for 1 mL per fish from a stock solution or from powder in Dulbecco&#39;s modified eagle medium (DMEM) with 1% v/v penicillin and streptomycin (see materials). Make aliquots (1 per fish) of 1 mL in 2 mL centrifuge tubes. Keep at 4 &#176;C until 30 min before the dissection step.</li>
	<li>For confocal microscopy/sample fixation, prepare 50 mL of fresh 4% paraformaldehyde (PFA) solution in PBS or thaw a stock solution from -20 &#176;C freezer in a fume hood. 
	CAUTION: PFA is toxic. Please read the material safety data sheet before working with it. Gloves and safety glasses should be worn, and always leave solutions inside&#160;a fume hood.</li>
</ol>
3. Preparing the Fluorescent Nanoparticle Suspension
<ol>
	<li>Label the protein nanoparticle with Atto-488 NHS ester (see Table of Materials) or an appropriate fluorescent dye according to the manufacturer&#39;s instructions.</li>
	<li>Resuspend the nanoparticles in 0.1 M sodium bicarbonate buffer at the concentration of 2 mg/mL.</li>
	<li>Dissolve the Atto 488 NHS ester in amine-free dimethyl sulfoxide (DMSO) at 2 mg/mL. Keep an aliquot of 10 &#181;L to check labeling efficiency (step 3.7&#8211;3.8).</li>
	<li>Mix the nanoparticles and the Atto 488 NHS ester at a molar ratio of 1:2 (protein:dye) by stirring in the dark.</li>
	<li>Spin down the labeled nanoparticles by centrifugation at 8,000 x g for 10 min at room temperature, remove the supernatant and keep it to check labeling efficiency (step 3.7&#8211;3.8).</li>
	<li>Wash the labeled nanoparticles by resuspending in 1 mL of 0.1 M sodium bicarbonate buffer by vortexing and pipetting up and down. Then discard the supernatant by centrifugation at 8,000 x g for 10 min at room temperature. Repeat step 3.6 for 5 times.</li>
	<li>Resuspend the pellet in 5 mL of 0.1 M sodium bicarbonate buffer in a 15 mL centrifuge tube and make aliquots of the fluorescent nanoparticle in 1.5 mL centrifuge tubes (30 aliquots). Spin down at 8,000 x g for 10 min at room temperature, discard the supernatant and store at -80 &#176;C protected from the light.</li>
	<li>Measure the labeling efficiency using a microvolume spectrophotometer.
	<ol>
		<li>Take 1 &#181;L of the original Atto 488 solution kept from step 3.3 and further dilute it in DMSO (e.g., 1:20 according to the volume ratio). This is the volume used to get the molar ratio of the protein nanoparticle and Atto 488 mix at step 3.4.</li>
		<li>Take 1 &#181;L of the saved supernatant from the labelling reaction in step 3.5. Measure the absorption (abs) at = 501 nm. The percentage of labelling is: 
		(<img alt="Equation 1" src="/files/ftp_upload/58366/58366eq1.jpg" /></li>
	</ol>
	</li>
	<li>Before the experiment, prepare the nanoparticle suspension at the desired concentration using 1x PBS solution.</li>
</ol>
4. Zebrafish Anesthetization and Oral Intubation
<ol>
	<li>Fast the fish (&#62;0.5 g) at least 48 h before the experiment to empty the intestine.</li>
	<li>Move the fish (12 fish) to the experimental tanks (6 L) one night before the experiment to allow acclimatization12.</li>
	<li>Vortex the nanoparticle solution well (e.g.,&#160;2,500 rpm and 30 s) and draw up the desired volume of nanoparticle suspension (e.g.,&#160;20&#8211;50 &#181;L) into the syringe attached to the protected needle.</li>
	<li>Place the fish in the aerated 150 mg/L MS-222 solution (see section 2) until they sink to the bottom of the tank and do not respond to a tail fin pinch; this process takes less than 5 min.</li>
	<li>Quickly transfer the anesthetized fish with a net to a wet plastic tray, orientate the animal horizontally to face the needle and immediately start the oral intubation.</li>
	<li>Carefully support the fish with one hand and open the mouth with the other hand using the protected needle. Gently insert the needle down the esophagus to about 1 cm from the mouth opening. 
	NOTE: The operator may feel a slight resistance when the end of the pipette tip has passed the gill. Take care not to angle the needle entry too much which may perforate the gill.</li>
	<li>Slowly inject the nanoparticle suspension to the fish. Make sure the suspension does not flow outward through the gills or mouth.</li>
	<li>Gently remove the needle and place the fish into the recovery tank (see section 2). Recovery usually takes within 1 min.</li>
	<li>Check the fish carefully for any abnormality (e.g., bleeding at the gills is a sign of perforation).</li>
	<li>Once the fish have recovered, return them to the experimental tanks.</li>
</ol>
5. Zebrafish Intestine Dissection
<ol>
	<li>After a specified period of time post intubation (e.g., 5 h and/or 24 h), place the fish using a net into 300 mg/L MS-222 solution for euthanasia (see section 2). Make sure the operculum stops moving and there is no tail pinch reflex. Five minutes is normally sufficient.</li>
	<li>Pick up the euthanized animal with a net and place it on a filter paper. 
	NOTE: The filter paper is very useful for removing the adhesive tissue along the intestine.</li>
	<li>Using sharp dissection scissors, make a semi-circular incision from the anus to the operculum and open incision using fine tweezers. Cut both ends of the intestine, take out all the internal organs, and place them on the filter paper. 
	CAUTION: Work quickly to reduce cell metabolism and death. 
	NOTE: Alternatively, remove the adhesive tissue in PBS and on ice.</li>
	<li>Separate the intestine from internal organs making sure to keep its orientation (anterior to posterior intestinal segment) and stretch it out. Usually, the anterior segment of intestine is wider than posterior segment. Take care to obtain all of the intestine when dissecting. 
	NOTE: The posterior end is quite fine and fragile in small fish and may break off, particularly in animals &#60;0.7 g.</li>
	<li>Roll the intestine on the filter paper with tweezers in order to detach the adhesive tissue from the intestine.</li>
	<li>Proceed to prepare the intestine for various downstream analyses (sections 6, 7 and 8).</li>
</ol>
6. Preparing Intestinal Cells for Cytometry
<ol>
	<li>Prepare in advance aliquots of 0.15% collagenase solution (see section 2.4). 
	NOTE: Aliquots should be at room temperature before proceeding.</li>
	<li>Optional:&#160;Continuing from step 5.5, slit open the intestine longitudinally and wash with 1x PBS.</li>
	<li>Using tweezers, place the intestine in the 2 mL centrifuge tube filled with 0.15% collagenase solution.</li>
	<li>Place the tubes on a vertical laboratory rotator for 1 h at room temperature in the dark.</li>
	<li>Place the intestine on a 100 &#181;m cell strainer supported over a 50 mL centrifuge tube. Break up the intestine with a 5 mL syringe plunger, washing 3 times with 1x PBS, collecting the flow through sample in the 50 mL centrifuge tube.</li>
	<li>Centrifuge the 50 mL centrifuge tube at 400 x g for 10 min, at 4 &#176;C.</li>
	<li>Carefully pipette off most of the supernatant while not losing the cells, some of which may be associated to the mucus.</li>
	<li>Resuspend the intestinal cells at the bottom of the centrifuge tube with 500 &#181;L of 1x PBS and keep on ice until cytometry analysis</li>
	<li>Filter samples through a 30 &#181;m cell filter into 5 mL round bottom tubes for cytometry.</li>
	<li>Set the parameters (e.g., the number of cells for analysis, the region of interest, voltage and compensation, selection of detectors) on a cytometer equipment (see materials).</li>
	<li>Immediately analyze the cells on a cytometer, following the instructions of use13.</li>
</ol>
7. Preparing Intestine Cryosections for Confocal Microscopy
<ol>
	<li>Fill the plastic mold (see Table of Materials) to half volume with optimum cutting temperature (O.C.T.) compound.</li>
	<li>Continuing from step 5.5 and immediately after the dissection, carefully place the intestine in the plastic mold. Make sure the intestine is totally embedded in the O.C.T. compound. If necessary, add more O.C.T. compound to the plastic mold. 
	NOTE: It is recommended to place the intestine with a &#34;Z&#34; shape in O.C.T. compound to easily follow its natural orientation.</li>
	<li>Place the plastic mold on dry ice until it goes opaque (less than a minute).</li>
	<li>Store the plastic mold at -80 &#176;C for long-term use or process immediately using the following procedure. 
	NOTE: The protocol can be paused here.</li>
	<li>Slice the frozen intestine into 10 &#181;m sections or appropriate thickness using a cryostat at -20 &#176;C.</li>
	<li>Collect the intestine section with a fine brush onto a slide.</li>
	<li>Immerse the slide in 4% PFA for 15 min at room temperature to fix the sample. 
	CAUTION: PFA is toxic. Please read the material safety data sheet before working with it. Gloves and safety glasses should be worn, and always leave solutions inside&#160;a fume hood.</li>
	<li>Wash the slide 3 times with 1x PBS, 10 min each.</li>
	<li>Add a drop of mounting medium and place a coverslip over the specimen. 
	NOTE: The protocol can be paused here.</li>
	<li>Observe the sample under a confocal microscope at appropriate magnification.</li>
</ol>
8. Preparing the Intestine for Real Time qPCR (RT-qPCR)
<ol>
	<li>Continuing from step 5.5, put the intestine in a cryogenic vial and rapidly freeze the intestine in liquid nitrogen and store at -80 &#176;C until use. 
	NOTE: The protocol can be paused here.</li>
	<li>For homogenization, add 200 &#181;L of 2% (v/v) chilled 1-Thioglycerol/Homogenization solution (see Table of Materials) or alternative homogenization solution to the intestine sample.</li>
	<li>Work quickly, homogenize the intestine sample on ice with a laboratory homogenizer at high speed (set at 25&#8211;30,000 rpm) until no visible tissue fragments remain. 3 times for&#160;5 s is usually sufficient.</li>
	<li>Isolate RNA using a commercial kit (see Table of Materials) according to the manufacturer&#39;s instructions14 or a suitable alternative method. As needed, store the RNA at -80 &#176;C for long-term use. 
	NOTE: The protocol can be paused here.</li>
	<li>Quantify the RNA concentration using a spectrophotometer15 and evaluate the quality using a RNA analyzer16.</li>
	<li>Prepare 1 &#181;g or an appropriate amount of cDNA using a cDNA synthesis kit according to the manufacturer&#39;s instructions. 
	NOTE: The protocol can be paused here. For qPCR analysis, please follow the MIQE guidelines17.</li>
	<li>Design appropriate primer pairs for the gene/s of interest.</li>
	<li>Select a suitable reference gene and analyze the expression of each gene by a RT-qPCR detection system using a commercial kit (see materials). 
	NOTE: For example, add 5 &#181;L of SYBR green supermix, 0.5 &#181;M primers, 2.5 &#181;L of diluted cDNA and 1.5 &#181;L of water in a final volume of 10 &#181;L for each well of qPCR plate.</li>
</ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+nanoparticles+and+Mycobacterium+marinum+across+the+intestinal+epithelium+in+zebrafish+and+the+role+of+the+mucosal+immune+system.">Translocation of nanoparticles and Mycobacterium marinum across the intestinal epithelium in zebrafish and the role of the mucosal immune system.</a> Developmental and Comparative Immunology. 67, 508-518 (2017).</li><li>Chen, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-Sized+mPEG-PLGA+Nanoparticles+of+Schisantherin+A+with+Sustained+Release+for+Enhanced+Brain+Uptake+and+Anti-Parkinsonian+Activity.">Small-Sized mPEG-PLGA Nanoparticles of Schisantherin A with Sustained Release for Enhanced Brain Uptake and Anti-Parkinsonian Activity.</a> ACS Applied Materials and Interfaces. 9 (11), 9516-9527 (2017).</li><li>Collymore, C., Rasmussen, S., Tolwani, R. 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The authors declare that no competing interests exist.

This protocol is an improvement of the previously described technique for oral intubation by Collymore et al.4 Our protocol describes in detail the oral intubation method and includes the preparation of the intestine for downstream analyses. Our method improves fish manipulation speed allowing one person to perform the whole protocol rapidly, without much variation between operators. A main difference of our protocol with the previous one is that we evaluate the success of an oral intubation experiment not only by observation of the animal for well-being (e.g., no bleeding) and for no leakage of the fluid administered, but also by checking the uptake of a bioactive nanoparticle in the intestine using downstream analysis (cytometry, confocal microscopy and qPCR). We show that intubated fluorescently labeled nanoparticles were found in the intestine.
On the practical side, the intubation equipment is cheap, precise and reusable: the basic device is made of a reusable 100 &#181;L glass (e.g., Hamilton) syringe coupled to a 31 G needle with a piece of silicon on the top. A cut sterile tip is placed over the silicon tube and can be changed for each individual administration. The syringe is reusable, and the thin needle allows intubating small fish with a high possibility of success. Moreover, the cut sterile tip could be placed directly over the needle without the silicon tube which allows the intubation equipment easier to be made in a laboratory. The administered bioactive compound is clearly visible, and a correct administration can be monitored easily. A critical step during the intubation procedure is the needle entry. It is important that the needle is not tilted or inserted too much to avoid perforating the gills. The mortality observed with this method is very low (approximately 7%) and depends on the size of the fish. Gill perforation is the most common cause of fish death and when it happens fish die within the first hour. Although fish of 0.5 g can be easily intubated the optimal fish size is around 1 g. Another difference with the method developed by Collymore et al.is that the fish are fasted for 48 h to be sure that gastrointestinal tract is empty. The whole intubation procedure can be done very quickly (30 animals/h) by one operator and importantly, the method is consistent between different operators4. The intubation method is easy to learn and does not require much practice to master.
The intestine dissection procedure must be properly performed to obtain good quality samples for cytometry, confocal and qPCR analyses. The critical step at this point is the dissection of the entire intestine; the posterior section is fragile and easy to lose. Once the intestine is dissected, it can be further processed for cytometry (2 h protocol), qPCR analysis (2 h protocol until total RNA isolation) or confocal microscopy (1 h to have samples ready for cryosection). It is very important to keep track of the orientation of the intestine, especially for confocal microscopy. Cytometry analysis requires rapid processing, and the procedure cannot be stopped before completion. Whereas, RNA isolation and samples prepared for cryosection can be properly stored and processed at any time. For cytometry, good quality samples without debris clumping the machine can be isolated with this method and fast analysis for the presence of fluorescent cells can be performed easily on individuals. RNA quality monitoring showed that high quality RNA can be isolated from intestine allowing analysis of individual fish by qPCR. Finally, the cryosection preparation for confocal microscopy provides important structural information about the protein nanoparticle uptake. Our methods thus provide a model to test the dynamics of oral prophylactics or other compounds in the intestine.
The limitations of our study are the size of the fish since we did not test administration in fish smaller than 0.5 g and the use of a chemical anesthetic to sedate the animals. Some authors use cold water (0&#8211;4 &#176;C) to anesthetize&#160;the zebrafish18 but in the context of animal welfare and European legal constraints we decided that MS-222 was the method of choice.
The zebrafish offers many advantages over other model systems including a complete set of gene data and the available transgenic lines. For immunologists, the transgenic lines (e.g., Tg mpx:GFP and Tg mpeg1:GFP) set a scene for the in vivo observation of immune cells such as macrophages and neutrophils19,20. Combining our oral intubation and downstream analysis with transgenic lines, could be ideal for identifying the cell types involving the uptake, transportation and processing of oral vaccines, nanoparticles and pathogens in fish.

The goal of this article is to describe in depth a straightforward method for oral intubation of zebrafish, along with useful associated downstream procedures. Oral intubation using zebrafish has become a practical model in the study of infectious disease dynamics, oral vaccine/immunostimulant, drug/nanoparticle uptake and efficacy, and intestinal mucosal immunity. For example, zebrafish oral intubation has been used in the study of Mycobacterium marinum and Mycobacterium peregrinum infection1. Lovmo et al. also successfully used this model to deliver nanoparticles and M. marinum to the gastro-intestinal tract of adult zebrafish2. In addition, Chen et al. used zebrafish oral intubation to show that drugs encapsulated by nanoparticles, when administered via the gastro-intestinal tract, were transported across the blood brain barrier3. These authors performed intubation based on the gauvage method described by Collymore et al.4 with some modifications. However, they did not provide a highly detailed protocol describing the oral intubation procedure. Here, we present a method for oral intubation of adult zebrafish building on Collymore et al.4 We further include the preparation of the intestine for relevant downstream analysis by cytometry, confocal microscopy and qPCR.
The intestine and particularly its mucosa is the first-line of defense against infection and the primary site of nutrient uptake5. When the epithelial cells and antigen-presenting cells within mucosal barriers perceive danger signals, an immediate innate immune response is triggered. Next, the highly specific adaptive immune response is established by T and B lymphocytes6,7. Development of oral vaccines is a current focus area in vaccinology. Such vaccines would be an effective tool to protect the organism at exposed sites due to the specific response of immune cells in the mucosa-associated lymphoid tissues (MALT)8,9. In aquaculture, mucosal vaccines have obvious advantages compared to injectable vaccines. They are practical for mass vaccination, less labor-intensive, are less stressful to the fish, and can be administered to young fish. Nevertheless, mucosal vaccine candidates must reach the second gut segment without being denatured in the oral environment. They also must cross mucosal barriers in order to gain access to antigen presenting cells (APCs) to induce local and/or systemic responses10. Hence, testing of the mucosal uptake achieved by candidate oral antigens and their delivery systems, as well as the immune response evoked, is essential in the development of oral vaccines.
In a biomedical context, developing a model to test biological effects of compounds after oral intubation is of growing interest. Many of the anatomical and physiological features of the intestine are conserved between bilaterian lineages, with mammals and bony fishes11. This oral intubation model connected to downstream analysis can be a tool to provide insights into human biology, as well as a testing ground for biologics or other compounds in vivo.
The oral intubation protocol can be performed by one operator, e.g., successfully administrating up to 50 &#181;L of the protein nanoparticle suspension&#160;to fish weighing 1 g, with a high survival rate. The procedure is simple to set up and quick; 30 fish can be intubated in 1 h. The protocol for intestine preparation is key to providing quality cell and tissue samples for subsequent analysis. Examples of downstream results are given which show the protocol&#39;s usefulness in obtaining data related to intestinal uptake and in isolating quality RNA for qPCR. The protocol would be of great use to those needing a suitable model to test the dynamics of oral prophylactics or other compounds in the intestine.

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			<td height="22" style="height:22px;width:289px;">Silicon tube</td>
			<td style="width:227px;">Dow Corning</td>
			<td style="width:295px;">508-001</td>
			<td style="width:460px;">0.30 mm inner diameter and 0.64 mm outer diameter</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Luer lock needle</td>
			<td style="width:227px;">Hamilton</td>
			<td style="width:295px;">7750-22</td>
			<td style="width:460px;">31 G, Kel-F Hub</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Luer lock syringe</td>
			<td style="width:227px;">Hamilton</td>
			<td style="width:295px;">81020/01</td>
			<td style="width:460px;">100 &#956;L, Kel-F Hub</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Filtered pipette tip</td>
			<td style="width:227px;">Nerbe Plus</td>
			<td style="width:295px;">07-613-8300</td>
			<td style="width:460px;">10 &#956;L</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">MS-222</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:295px;">E10521</td>
			<td style="width:460px;">powder</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">10x PBS</td>
			<td style="width:227px;">Sigma Aldrich</td>
			<td style="width:295px;">P5493</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Filter paper&#160;</td>
			<td style="width:227px;">Filter-Lab</td>
			<td style="width:295px;">RM14034252</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Collagenase</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">17104019</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">DMEM&#160;</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">31966</td>
			<td style="width:460px;">Dulbecco&#39;s modified eagle medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Penicillin and streptomycin</td>
			<td style="width:227px;">Gibco</td>
			<td style="width:295px;">15240</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cell strainer</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:295px;">352360</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">CellTrics filters&#160;</td>
			<td style="width:227px;">Sysmex Partec</td>
			<td style="width:295px;">04-004-2326 (Wolflabs)</td>
			<td style="width:460px;">30 &#181;m mesh size filters with 2 mL reservoir</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:227px;">SAKURA</td>
			<td style="width:295px;">4583</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Plastic molds for cryosections</td>
			<td>SAKURA</td>
			<td>4557</td>
			<td>Disposable Vinyl molds. 25 mm x 20 mm x 5 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Slide</td>
			<td style="width:227px;">Thermo Scientific</td>
			<td style="width:295px;">10149870</td>
			<td style="width:460px;">SuperFrost Plus slide</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cover glasses</td>
			<td style="width:227px;">Labbox&#160;</td>
			<td style="width:295px;">COVN-024-200</td>
			<td style="width:460px;">24&#180;24 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Paraformaldehyde (PFA)</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">158127</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Atto-488 NHS ester</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">41698</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Sodium bicarbonate</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">S5761</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">DMSO</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">D8418</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Maxwell RSC simplyRNA Tissue Kit</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">AS1340</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:289px;">1-Thioglycerol/Homogenization solution</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">Inside of Maxwell RSC simplyRNA Tissue Kit</td>
			<td style="width:460px;">adding 20 &#956;l 1-Thioglycerol to 1 ml homogenization solution (2%)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">vertical laboratory rotator&#160;</td>
			<td style="width:227px;">Suministros Grupo Esper</td>
			<td style="width:295px;">10000-01062</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Cryostat</td>
			<td style="width:227px;">Leica&#160;</td>
			<td style="width:295px;">CM3050S</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Homogenizer</td>
			<td style="width:227px;">KINEMATICA</td>
			<td style="width:295px;">Polytron PT1600E</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Flow cytometer&#160;</td>
			<td style="width:227px;">Becton Dickinson</td>
			<td style="width:295px;">FACS Canto</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">5 mL round bottom tube</td>
			<td style="width:227px;">Falcon</td>
			<td style="width:295px;">352058</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Confocal microscope</td>
			<td style="width:227px;">Leica</td>
			<td style="width:295px;">SP5</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Fume Hood</td>
			<td style="width:227px;">Kottermann</td>
			<td style="width:295px;">2-447 BST</td>
			<td style="width:460px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Nanodrop 1000</td>
			<td style="width:227px;">Thermo Fisher Scientific</td>
			<td style="width:295px;">ND-1000</td>
			<td style="width:460px;">Spectrophotometer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Agilent 2100 Bioanalyzer System</td>
			<td style="width:227px;">Agilent</td>
			<td style="width:295px;">G2939A</td>
			<td>RNA bioanalyzer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Maxwell Instrument</td>
			<td style="width:227px;">Promega</td>
			<td style="width:295px;">AS4500&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">iScript cDNA synthesis kit&#160;</td>
			<td style="width:227px;">Bio-rad</td>
			<td style="width:295px;">1708891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:289px;">CFX384 Real-Time PCR&#160;Detection System</td>
			<td style="width:227px;">Bio-Rad</td>
			<td style="width:295px;">1855485</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:289px;">iTaq universal SYBR Green Supermix kit</td>
			<td style="width:227px;">Bio-rad</td>
			<td style="width:295px;">172-5120</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Water&#160;</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">W4502</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Cryogenic vial&#160;</td>
			<td style="width:227px;">Thermo Fisher Scientific</td>
			<td style="width:295px;">375418</td>
			<td>CryoTube vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:289px;">Mounting medium</td>
			<td style="width:227px;">Sigma-Aldrich</td>
			<td style="width:295px;">F6057</td>
			<td>Fluoroshield with DAPI</td>
		</tr>
	</tbody>
</table>

oral intubation of adult zebrafish: a model for evaluating intestinal uptake of bioactive compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

Zebrafish (average weight: 1.03 &#177; 0.16 g) of mixed sex were successfully intubated with different recombinant protein nanoparticles (bacterial inclusion bodies) using our home-made oral intubation device (Figure 1). We have successfully performed the oral intubation and achieved a low average percentage mortality (6.8%) (Table 1). Zebrafish were either intubated with 30 &#181;L or 50 &#181;L of nanoparticle suspensions and the mortality was calculated within 24 h post intubation. The experiments were performed by two operators, R had less experience working with zebrafish oral intubation than J. The results showed that even a new operator could independently perform the oral intubation experiment and easily achieve a high survival rate by this protocol. From our experience, the optimal fish size is 1 g, but we have successfully intubated fish as small as 0.5 g.
To better understand whether the fluorescent nanoparticles IBsTNF&#945; (a recombinant cytokine protein nanostructured as inclusion bodies) were delivered to zebrafish by our method and taken up by the zebrafish intestine or not, we performed cytometry analysis. The nanoparticles (100 &#181;g/fish, 50 &#181;L) and PBS (control) were orally administered (by intubation) to zebrafish and the intestine was dissected at 5 h and 24 h post intubation. Total intestinal cells were prepared by step 6 and analyzed by detecting the fluorescence emission signal. The representative histograms of fluorescence intensity and the dot plots of fluorescent cell percentage are shown in Figure 2. The density of fluorescent cells is clearly higher in nanoparticle intubated group compared to the control group in both 5 h and 24 h (Figure 2A). The percentages of fluorescent cells are significantly higher in both 5 h (46.3%) and 24 h (43.0%) of nanoparticle intubated groups (Figure 2B).
To further study which part of the intestinal layer is involved in the nanoparticle uptake, we performed confocal microscopy analysis. The nanoparticles (20 &#181;g/fish, 50 &#181;L) and PBS (control) were orally intubated to zebrafish and the intestine was dissected at 5 h post intubation. The intestine sections were prepared by a frozen tissue method according to step 7 (Figure 3A). The confocal images of fluorescent nanoparticles in the intestine are shown in Figure 3B. The fluorescent nanoparticles were found in zebrafish intestine. We observed the fluorescence in the epithelial cells, lamina propria, and muscle cells.
To verify whether we could extract high quality RNA by our protocol, we analyzed the RNA extracted from the intestine with a bioanalyzer (also referred to as RNA analyzer here). Zebrafish were intubated orally with PBS (50 &#181;L) or the nanoparticles (20 &#181;g/fish, 50 &#181;L). The intestines were dissected for RNA extraction at 24 h post intubation. We selected seven RNA samples to test with the analyzer. We found that all the tested RNA samples have high RNA integrity numbers ranging from 7.9 to 8.9 (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58366/58366fig1.jpg" /> 
Figure 1: Oral intubation device. (A) Image of a 31 G Luer lock needle fixed on a 100 &#181;L syringe with the silicon tube and pipette tip end covering the tip of the needle. (B) An enlarged image of the needle part. The black arrow indicates where the pipette tip end exceeds the tip of the needle. <a href="https://www.jove.com/files/ftp_upload/58366/58366fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58366/58366fig2.jpg" /> 
Figure 2: Flow cytometry analysis of fluorescent nanoparticles in zebrafish intestine via oral intubation. Zebrafish were treated with PBS or fluorescent IBsTNF&#945; (100 &#181;g) for 5 h and 24 h, respectively. (A) Representative histograms of fluorescence intensity. (B) Dot plots graph of fluorescent cells percentage. Each green dot represents the percentage of fluorescent cells in one individual, n &#8805;4. Data represent mean &#177; standard error of the mean (SEM). Differences were analyzed using one-way ANOVA. Significant differences with respect to control (**, p &#60;0.01) <a href="https://www.jove.com/files/ftp_upload/58366/58366fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58366/58366fig3.jpg" /> 
Figure 3: Images&#160;of confocal microscopy analysis. Fish were orally intubated with PBS or 20 &#181;g/fish fluorescent nanoparticles. The intestine was dissected at 5 h post intubation. (A) Zebrafish intestine embedded in OCT compounds. The intestine was placed with the natural orientation of a &#34;Z&#34; shape (a: anterior end; p: posterior end). (B) Confocal microscopy images of zebrafish intestine. The white arrows show that the fluorescent nanoparticles are taken up in the intestinal mucosa. <a href="https://www.jove.com/files/ftp_upload/58366/58366fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58366/58366fig4.jpg" /> 
Figure 4: RNA analyzer virtual gel image showing RNA extracted from 7 zebrafish intestines sampled post intubation. Sample number 1 and 2 are PBS intubated groups and sample number 3 to 7 are nanoparticle intubated groups. RNA integrity numbers (RIN) are given at the bottom ranging from 7.9 to 8.9. <a href="https://www.jove.com/files/ftp_upload/58366/58366fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Operator</td>
			<td>Volume</td>
			<td># fish intubated</td>
			<td>Mean weight 
			(g &#177; SD)</td>
			<td># deaths</td>
			<td>Mortality (%)</td>
		</tr>
		<tr>
			<td>R</td>
			<td>30 &#181;L&#160;</td>
			<td>22</td>
			<td>0.88 &#177; 0.14</td>
			<td>3</td>
			<td>13.6</td>
		</tr>
		<tr>
			<td>R</td>
			<td>30 &#181;L</td>
			<td>17</td>
			<td>0.93 &#177; 0.19</td>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>J</td>
			<td>50 &#181;L</td>
			<td>19</td>
			<td>1.23 &#177; 0.31</td>
			<td>1</td>
			<td>5.2</td>
		</tr>
		<tr>
			<td>J</td>
			<td>50 &#181;L</td>
			<td>30</td>
			<td>1.08 &#177; 0.40</td>
			<td>2</td>
			<td>6.6</td>
		</tr>
		<tr>
			<td>Total</td>
			<td></td>
			<td>88</td>
			<td>1.03 &#177; 0.16</td>
			<td>6</td>
			<td>6.8</td>
		</tr>
		<tr>
			<td colspan="6">SD: standard deviation of the mean</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of zebrafish mortality caused by two operators using the protocol. The operator identification code, the intubated volume, the fish number, and the fish average weight in grams (g) are shown.

Watch this Scientific Journal Video about Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds at JoVE.com

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

oral-intubation-adult-zebrafish-model-for-evaluating-intestinal

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

7.7K Views.  Universitat Autònoma de Barcelona. The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It is relevant for testing the intestinal dynamics of oral prophylactics.

Video: Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

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 ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...