Name: oral intubation of adult zebrafish: a model for evaluating intestinal uptake of bioactive compounds
Uploaded: 2018-09-27T15:00:00
Description: Watch this Scientific Journal Video about Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds at JoVE.com

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...

<ol>
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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 intubat...

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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	<tbody>
		<tr height="22">
			<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 ...

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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 ...

This method can help answer key questions in the mucosa vaccinology field, such as how intestinal mucosa uptake biological compounds and how such biologicals modulate the immune system at local site after intubation. The main advantage of this technique is that it's simple to set up, quick, and shows low mortality rates. This method can provide insight into mucosal vaccinology.It can also be applied to other compounds, such as chemicals, toxics, or pathogens. Visual demonstration of this method is critical as in the oral intubation and intestinal dissection steps, you need to be careful because in intubation you could injure the zebrafish and cause death. In dissection, the end of the intestine is very fine and fragile.Place approximately one centimeter of fine silicone tubing on a 31-gauge Luer-lock needle. Cut a sterile 10-microliter filter pipette tip, take the finer end, and place it over the silicone tube as a sheath. To begin, attach the needle apparatus to a 100-microliter Luer-lock syringe.First, move the fish to the experimental tanks one night prior to the experiment. Vortex the prepared nanoparticle solution well, and draw up the desired volume of the suspension. After safely anesthetizing the fish, use a net to quickly transfer the fish to a wet plastic tray.Orient the animal horizontally to face the needle. Carefully support the fish with one hand, and open the mouth with the protected needle. Then, gently insert the needle down the esophagus to about one centimeter from the mouth opening.It's difficult to see how far the needle should go, and this is a matter of practice. You will feel the needle as it finds a stop, and then by manipulating the needle, you'll find the correct angle to insert the device a little more. Slowly inject the nanoparticle suspension into the fish, ensuring the suspension does not flow outward through the gills or the mouth.After this, gently remove the needle, and place the fish into a recovery tank. While the fish recovers, monitor the fish for any abnormalities. Once the fish has recovered, return it to one of the experimental tanks.After euthanizing the fish according to the text protocol, place the fish on a piece of filter paper. Using sharp dissection scissors, make a semi-circular incision from the anus to the operculum, and open the incision with fine tweezers. Then, cut both ends of the intestine, take out all of the internal organs, and place them on the filter paper.Next, separate the intestine from the internal organs, and stretch it out, taking care to obtain all of the intestine. Finally, use tweezers to roll the intestine on the filter paper to detach the adhesive tissue. Using this protocol, zebrafish of mixed sex were successfully intubated with different recombinant protein nanoparticles.Using the homemade oral intubation device, a low average percentage of mortality was achieved. To evaluate the delivery of the fluorescent nanoparticles inclusion bodies made with recombinant TNF alpha, a cytometry analysis was performed. The total amount of fluorescent cells was significantly higher in the nanoparticle-intubated group than in the control group both five and 24 hours after intubation.The intubation equipment is inexpensive, precise, and reusable. The whole intubation procedure can be done very quickly by one operator, and, importantly, the method is consistent between different operators. The combination of our intubation method with downstream analysis could be ideal for oral vaccine development.

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

Research

JoVE Journal

Immunology and Infection

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&amp;#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 ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...