Name: a ligated intestinal loop model in anesthetized specific pathogen free chickens to study clostridium perfringens virulence
Uploaded: 2018-10-11T12:30:00
Description: Watch this Scientific Journal Video about A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence at JoVE.com

This work was supported by the Chair in Poultry Research (M. Boulianne) from the Faculty of Veterinary Medicine of the Universit&#233; de Montr&#233;al. Drs. Boulianne and Parent developed the model. Drs. Boulianne and Parent performed the surgeries. Dr. Burns assisted in developing the anesthesia protocol. Dr. Parent edited the video and wrote the manuscript. Drs Burns, Desrochers and Boulianne edited the manuscript.

All procedures with live animal use were authorized by the Ethical Committee of the Faculty of Veterinary Medicine, Universit&#233; de Montr&#233;al (C&#201;UA, &#39;Comit&#233; d&#39;&#233;thique de l&#39;utilisation des Animaux&#39;).
1. Considerations Before Surgery
<ol>
	<li>Select 10 weeks old specific pathogen free (SPF) leghorn chickens for the surgery. 
	NOTE: Their weight must be between 1.0 and 1.2 kg.</li>
	<li>Withdraw the feed 12 h prior to the procedures to empty the intes...

<ol>
	<li>Vitale, A., Chiarotti, F., Alleva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+animal+models+in+disease+research.">The use of animal models in disease research.</a> Rare diseases and orphan drugs. 2 (1), 1-4 (2015).</li><li>Uzal, F. A., 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=Animal+models+to+study+the+pathogenesis+of+human+and+animal+Clostridium+perfringens+infections.">Animal models to study the pathogenesis of human and animal Clostridium perfringens infections.</a> Veterinary Microbiology. , (2015).</li><li>Bernier, G., Phaneuf, J. B., Filion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotic+enteritis+in+broiler+chickens.+III.+Study+of+the+factors+favoring+the+multiplication+of+Clostridium+perfringens+and+the+experimental+transmission+of+the+disease.">Necrotic enteritis in broiler chickens. III. Study of the factors favoring the multiplication of Clostridium perfringens and the experimental transmission of the disease.</a> Canadian Journal of Comparative Medicine. 41 (1), 112-116 (1977).</li><li>Al-Sheikhly, F., Truscott, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+of+necrotic+enteritis+of+chickens+following+infusion+of+broth+cultures+of+Clostridium+perfringens+into+the+duodenum.">The pathology of necrotic enteritis of chickens following infusion of broth cultures of Clostridium perfringens into the duodenum.</a> Avian Diseases. 21 (2), 230-240 (1977).</li><li>Wicker, D. L., Iscrigg, W. N., Trammell, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+and+prevention+of+necrotic+enteritis+in+broilers+with+zinc+bacitracin.">The control and prevention of necrotic enteritis in broilers with zinc bacitracin.</a> Poultry Science. 56 (4), 1229-1231 (1977).</li><li>Keyburn, A. L., 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=NetB,+a+new+toxin+that+is+associated+with+avian+necrotic+enteritis+caused+by+Clostridium+perfringens.">NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens.</a> PLoS Pathog. 4 (2), e26 (2008).</li><li>Timbermont, L., 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=Origin+of+Clostridium+perfringens+isolates+determines+the+ability+to+induce+necrotic+enteritis+in+broilers.">Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers.</a> Comp Immunol Microbiol Infect Dis. 32 (6), 503-512 (2009).</li><li>Paradis, M. A., 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=Efficacy+of+avilamycin+for+the+prevention+of+necrotic+enteritis+caused+by+a+pathogenic+strain+of+Clostridium+perfringens+in+broiler+chickens.">Efficacy of avilamycin for the prevention of necrotic enteritis caused by a pathogenic strain of Clostridium perfringens in broiler chickens.</a> Avian Pathol. 45 (3), 365-369 (2016).</li><li>Valgaeren, B., 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=Lesion+development+in+a+new+intestinal+loop+model+indicates+the+involvement+of+a+shared+Clostridium+perfringens+virulence+factor+in+haemorrhagic+enteritis+in+calves.">Lesion development in a new intestinal loop model indicates the involvement of a shared Clostridium perfringens virulence factor in haemorrhagic enteritis in calves.</a> Journal of Comparative Pathology. 149 (1), 103-112 (2013).</li><li>Goossens, E., 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=The+C-terminal+domain+of+Clostridium+perfringens+alpha+toxin+as+a+vaccine+candidate+against+bovine+necrohemorrhagic+enteritis.">The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis.</a> Vet Res. 47 (1), 52 (2016).</li><li>Goossens, E., 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=Toxin-neutralizing+antibodies+protect+against+Clostridium+perfringens-induced+necrosis+in+an+intestinal+loop+model+for+bovine+necrohemorrhagic+enteritis.">Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis.</a> BMC Vet Res. 12 (1), 101 (2016).</li><li>Parent, E., Archambault, M., Charlebois, A., Bernier-Lachance, J., Boulianne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chicken+intestinal+ligated+loop+model+to+study+the+virulence+of+Clostridium+perfringens+isolates+recovered+from+antibiotic-free+chicken+flocks.">A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks.</a> Avian Pathol. 46 (2), 138-149 (2017).</li><li>Janvilisri, 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=Temporal+differential+proteomes+of+Clostridium+difficile+in+the+pig+ileal-ligated+loop+model.">Temporal differential proteomes of Clostridium difficile in the pig ileal-ligated loop model.</a> PLoS One. 7 (9), e45608 (2012).</li><li>Aabo, S., 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=Development+of+an+in+vivo+model+for+study+of+intestinal+invasion+by+Salmonella+enterica+in+chickens.">Development of an in vivo model for study of intestinal invasion by Salmonella enterica in chickens.</a> Infection and Immunity. 68 (12), 7122-7125 (2000).</li><li>Meurens, F., 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=Early+immune+response+following+Salmonella+enterica+subspecies+enterica+serovar+Typhimurium+infection+in+porcine+jejunal+gut+loops.">Early immune response following Salmonella enterica subspecies enterica serovar Typhimurium infection in porcine jejunal gut loops.</a> Vet Res. 40 (1), 5 (2009).</li><li>Gerdts, V., 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=Multiple+intestinal+'loops'+provide+an+in+vivo+model+to+analyse+multiple+mucosal+immune+responses.">Multiple intestinal 'loops' provide an in vivo model to analyse multiple mucosal immune responses.</a> J Immunol Methods. 256 (1-2), 19-33 (2001).</li><li>Wade, B., 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=The+adherent+abilities+of+Clostridium+perfringens+strains+are+critical+for+the+pathogenesis+of+avian+necrotic+enteritis.">The adherent abilities of Clostridium perfringens strains are critical for the pathogenesis of avian necrotic enteritis.</a> Vet Microbiol. 197, 53-61 (2016).</li><li>Cooper, K. K., 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=Virulence+for+chickens+of+Clostridium+perfringens+isolated+from+poultry+and+other+sources.">Virulence for chickens of Clostridium perfringens isolated from poultry and other sources.</a> Anaerobe. 16 (3), 289-292 (2010).</li><li>Cooper, K. K., Songer, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Clostridium+perfringens+in+an+experimental+model+of+poultry+necrotic+enteritis.">Virulence of Clostridium perfringens in an experimental model of poultry necrotic enteritis.</a> Veterinary Microbiology. 142 (3-4), 323-328 (2010).</li><li>Chalmers, G., Bruce, H. L., Toole, D. L., Barnum, D. A., Boerlin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrotic+enteritis+potential+in+a+model+system+using+Clostridium+perfringens+isolated+from+field+outbreaks.">Necrotic enteritis potential in a model system using Clostridium perfringens isolated from field outbreaks.</a> Avian Diseases. 51 (4), 834-839 (2007).</li></ol>

Intestinal loops models have been described in numerous species to study host-pathogen interaction and pathogenesis of diseases caused by various intestinal pathogens, such as Clostridium perfringens, Clostridium difficile and Salmonella enterica7,9,13,14,15. It has also been used to analyze the mucosal immune response<sup class="xre...

The use of animal models to study infectious diseases affecting humans and animals is a central tool in assessing virulence factors regulating the pathogenesis of a specific condition as well as to elaborate strategies to prevent or cure diseases1. Despite numerous advantages of using animal models to study various diseases, ethical concerns regarding this practice are arising. Researchers need to minimize animal use while obtaining significant and valid results. The concept of the 3 Rs principles (Replace, Reduce and Refine) was elaborated to ensure that welfare issues were addressed in such trials. In chicken necrotic enteritis studies, in vivo chicken models are used to investigate the etiology, prevention, and treatments of this condition2,3,4,5. Pathogenic strains of C. perfringens carrying specific virulence factors, such as the NetB toxin6, are administered to cause necrotic enteritis in chickens7. The replacement principle is, therefore, difficult to achieve in such case as these virulence factors may not be as critical in other animal species. Most models for necrotic enteritis in chickens use a combination of risk factors, such as coccidiosis and altered diet to induce necrotic enteritis followed by gavage of broth culture containing large numbers of C. perfringens2. These models will induce the disease in a large number of chickens, and mortality can be important in such trials8, thus raising concerns about the reduction and refinement principles in animal research.
Intestinal ligated loops models are a desirable alternative for the study of diseases induced by intestinal pathogens with respect to the principle reduction, and refinement. In these models, intestinal segments called &#39;loops&#39; are created by placing ligatures along the intestinal tract to form independent and hermetic compartments where pathogens can be injected alone9 or with other molecules, such as vaccine candidates10,11. The pathogens of interest are placed in close contact with the intestinal cells and after a few hours of infection time, intestinal samples can be recovered for further analysis. This allows the use of multiple treatment and control groups in the same animal. Statistical analysis can be performed with repeated measures models, which increases the power of discrimination between groups and reduces the number of necessary chickens compared to oral gavage trials. Also, surgical procedures and subsequent infection times are performed under continuous general anesthesia and analgesia, hence minimizing the animal pain. Closed loop ligations are an ideal template for reducing host numbers and creating a more humane system in animal research.
Intestinal ligated loops models are well described in various species, such as calves, rabbits and mice2,9, but poorly described in chickens7. For an optimal use of this surgical model, proper technique and execution are essential for the creation of ligated intestinal loops to avoid damages to the intestinal integrity. The goal of this manuscript is to describe a step by step method in the creation of multiple intestinal loops in a chicken model. This technique is limited by the surgeon&#39;s skill and experience, as accurate procedures are essential for the success of the project.

<table>
	<tbody>
		<tr>
			<td>EZ-Scrub 747</td>
			<td>Becton Dickinson and Company</td>
			<td></td>
			<td>4% chlorhexidine gluconate detergent impregnated sterile brush. No catalog number available. Web address: https://www.bd.com/en-us/offerings/capabilities/infection-prevention/surgical-hand-scrubs/ez-scrub-preoperative-surgical-scrub-brushes&#160;</td>
		</tr>
		<tr>
			<td>Isopropyl alcohol 70% USP 4 L</td>
			<td>Commercial isopropyl alcohol</td>
			<td>P016IP70</td>
			<td>Web address: http://www.comalc.com/products/</td>
		</tr>
		<tr>
			<td>Chlorexidine</td>
			<td>Sigma-Aldrich</td>
			<td>282227-1G</td>
			<td>Chlorhexidine gluconate solution, must be diluted to 4%. Web address: http://www.sigmaaldrich.com/catalog/product/aldrich/282227?lang=fr&#38;region=CA&#38;gclid=EAIaIQobChMI8vHr4_OY1wIV3rrACh2ZWQKuEAAYAiAAEgLKx_D_BwE</td>
		</tr>
		<tr>
			<td>Surgical Drape Small (27 inch x 24 inch) with 3 x 6 inch Fenestration</td>
			<td>Veterinary Specialty Products</td>
			<td>32724</td>
			<td>Web address: http://www.vetspecialtyproducts.com/index.cfm?fuseaction=ecommercecatalog.detail&#38;productgroup_id=15</td>
		</tr>
		<tr>
			<td>Scalpel blades #3</td>
			<td>Swann-Morton</td>
			<td>301</td>
			<td>Web address: https://www.swann-morton.com/product/16.php</td>
		</tr>
		<tr>
			<td>Sterile saline NaCl 0.9%</td>
			<td>Sigma-Aldrich</td>
			<td>S8776-100ML</td>
			<td>Web address: http://www.sigmaaldrich.com/catalog/product/sigma/s8776?lang=fr&#38;region=CA</td>
		</tr>
		<tr>
			<td>Vicryl 3-0</td>
			<td>Ethicon</td>
			<td>D9003</td>
			<td>Polyglactin multifilament absorbable suture material. Web address: http://www.ethicon.com/healthcare-professionals/products/wound-closure/absorbable-sutures/coated-vicryl-polyglactin-910-suture</td>
		</tr>
		<tr>
			<td>Syringe 1 ml with 26G needle</td>
			<td>Becton Dickinson and Company</td>
			<td>329652</td>
			<td>Web address: https://www.bd.com/en-us/offerings/capabilities/diabetes-care/insulin-syringes/bd-1-ml-conventional-insulin-syringes</td>
		</tr>
		<tr>
			<td>Brain Heart Infusion</td>
			<td>Sigma-Aldrich</td>
			<td>1104930500</td>
			<td>Web address: http://www.sigmaaldrich.com/catalog/product/mm/110493?lang=fr&#38;region=CA&#38;cm_sp=Insite-_-prodRecCold_xorders-_-prodRecCold2-1</td>
		</tr>
		<tr>
			<td>Eppendorf tube</td>
			<td>Sigma-Aldrich</td>
			<td>T9661-500EA</td>
			<td>Microcentrifuge tube. Web address: http://www.sigmaaldrich.com/catalog/product/sigma/t9661?lang=fr&#38;region=CA&#38;gclid=EAIaIQobChMI-YizzfiY1wIVRkCGCh30SQjuEAAYAiAAEgLK5fD_BwE</td>
		</tr>
		<tr>
			<td>Formalin solution, buffered neutral, 10%</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128-4L</td>
			<td>Ratio tissue : formalin of 1: 10 for adequate fixation. Web address: http://www.sigmaaldrich.com/catalog/product/sigma/ht501128?lang=fr&#38;region=CA</td>
		</tr>
		<tr>
			<td>Clostridium perfringens strains</td>
			<td>Universit&#233; de Montr&#233;al, Chaire en recherche avicole</td>
			<td>N/A</td>
			<td>Specific to each laboratory, available upon request to correspondant author</td>
		</tr>
	</tbody>
</table>

a ligated intestinal loop model in anesthetized specific pathogen free chickens to study clostridium perfringens virulence

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as coccidiosis and diet, with gavage or administration via the feed using Clostridium perfringens. In these models, the comparison of multiple C. perfringens strains for virulence studies requires a large number of hosts to obtain significant results. Mortality during the course of the study can be high depending on the experimental model, hence raising ethical concerns regarding animal welfare in research. The development of new infection models requiring fewer animals to study pathogenesis, yet providing statistically significant and valid results, is important in reducing animal use in research. Intestinal ligated loop models have been used to study clostridial infections in various species such as mice, rabbits and calves. Following surgical procedures to create ligated loop segments, C. perfringens strains are injected directly into the loops to establish a close contact between the bacteria and the intestinal mucosa. Samples of the small intestine and luminal contents are taken at the termination of the procedures after a few hours. Multiple bacterial strains can be inoculated in each animal, hence reducing the number of required subjects in the experiments. Also, procedures are performed under general anesthesia to reduce animal pain. In chickens, this model would be more appropriate than oral administration to compare C. perfringens strain pathogenicity because fewer animals are needed, no predisposing factors are required to induce the disease, and pain is controlled by analgesics. The intestinal ligated loop model is poorly described in chickens and standardization is essential for its optimal use. This manuscript provides all the necessary steps to create numerous intestinal ligated loops in chickens and brings information on the critical points to obtain valid results.

A schematic of the 9 intestinal loops and 8 interloops is shown in Figure 1. In this model, a total of 9 loops and 8 interloops are created with simple ligatures. A loop consists of proximal and distal ligatures spaced by 2 cm measured from the proximal ligature. Two adjacent loops are separated by an interloop of 1 cm. To decrease the possible risk of cross-contamination between loops by leakage from a ligature, an interloop ligature is placed mid-way from t...

Watch this Scientific Journal Video about A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence at JoVE.com

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Here we present a protocol to surgically create &#39;intestinal ligated loops&#39; in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains&#39; virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as ...

This method can help answer key questions in the field of Chicken Necrotic Enteritis research. Such as comparing the virulence of specific Clostridium perfringens strains causing this disease. The main advantage of this technique is that it remarkably reduces the number of chickens usually necessary for similar ambivore experiments.While this method can provide insight into virulence potential of Clostridium perfringens strains, it can also be applied to other intestinal pathogens such as Clostridium difficile, or Salmonella enterica. Generally, individuals new to this method will struggle because surgical procedures needs specific training and shall only be performed by those who have acquired skills such as suturing, an techniques prior to the experiment. After anesthesizing a 10 week old specific pathogen free leghorn chicken, set up the monitors for vital measurements and start manually removing the feathers from the abdomen by gentle traction.Next, scrub the surgical site using Chlorhexidine gluconate with a soft bristle brush. Gently scrub the skin from the center to the periphery for five minutes without reversing course. Then, perform three alternating passages on the surgical site with Chlorhexidine gluconate solution and Isopropyl alcohol using sterile gauzes.Again, go from the center to the periphery, then, drape the chicken. Begin by opening the drape above the surgical site, then make an L-shaped low midline skin incision with a number three scalpel, starting one centimeter cuddle to the sternum, and ending at one centimeter cranial to the cloicha. Next, continue the L-shaped incision perpendicular to the first by doing a second incision starting at the cuddle end of the first incision and continue five centimeters to the left side of the abdomen by following the pelvis line.This opening will allow easy extraction of the intestines. Next, open the peritoneum and abdominal muscles using the same L-shaped pattern to access the abdominal cavity. The air sacs will now be exposed and must be kept moist using saline soaked gauze.Next, insert a Snook spay hook into the abdominal cavity by following the left abdominal wall and working the hook around the abdominal air sacs to extract the intestines. Then, using dry gauze, continue gently exteriorizing the intestines to expose the Jejunum. Spread out the intestines and keep them moist with frequent spraying with Saline and by covering them with Saline soaked gauze.It is important to be very gentle while taking out intestines because excessive tension can rupture the mesenteric vessels which may lead to intestinal ischemic lesions and greatly compromise the outcome of the procedure. After exteriorizing the intestines, make a series of loops in the intestines beginning in the Jejunum. Each loop can be used for different treatment.In the proximal Jejunum, place a simple ligature with a Polyglutamine Multifilament synthetic absorbable material while avoiding the ligature of major mesenteric vessels. Next, place a distal ligature two centimeters away from the proximal ligature to complete the first loop. All proximal and distal ligatures should be spaced by about two centimeters.Each pair of ligatures makes a loop, which is a hermetic segment. Next, create an interloop or a segment of intestine between the loops. Place a simple ligature half a centimeter aborely to the distal ligature of the first loop.This ligature is at the middle of the interloop and will decrease possible cross contamination between loops. Now, continue the pattern of creating another loop half a centimeter aborely from the interloop ligature. Here, nine loops and eight interloops are created covering 26 centimeters of intestine.The number of loops can be adjusted as needed. Next, proceed with injecting strain of Clostridium perfringens into the loops. Into the anti-mesenteric side of the loop, use a 26 gauge needle at a 45 degree angle to inject 0.2 milliliters of BHI solution, containing one million colony forming units of bacteria in the midlog growth phase.In this situation, with nine loops, five different strains are injected in alternating loops with the intervening loops injected with vehicle alone as negative controls. After injecting the pathogens, gently replace the intestinal tract to the dorsal area of the abdominal cavity underneath the air sacs. Then, close the abdominal cavity.Suture the peritoneum and abdominal muscles along the pelvis with a simple continuous suture pattern using a Polyglutamine Multifilament synthetic absorbable material. Continue by suturing the peritoneum and abdominal muscles from the sternum to the cloiche using simple continuous sutures. Next, suture the skin close using a Polyglutamine Multifilament synthetic material and a simple continuous pattern.First, suture along the pelvis, then suture from the sternum to the cloiche. After closing the incisions, keep the chicken under general anesthesia with proper use of analgesics to minimize animal pain during the infection time. Continue anesthetic monitoring to ensure an adequate anesthesia level.After the desired amount of infection time, such as seven hours in the present experiment, euthanize the bird and collect the intestinal tissues. Using a number three scalpel, cut out a half to one centimeter loop section. Then, transfer the section to 10%Formalin for overnight fixation and further histopathological analysis.Then, use the same scalpel blade on the same loop to cut out the remainder of the loop and transfer that tissue to a microfused tube for isolation of the bacteria. Repeat this procedure for each loop using a new scalpel blade each time. Seven hours after injection, an intestinal loop injected with a control sterile medium has an intact mucosal brush border with no necrosis.Mild congestion in the intestinal layers of the segments is not uncommon. Seven hours following injection with the pathogen, Clostridium perfringens, villi tips are eroded with necrotic entry sites surrounding the tips. And then necrotic material surrounding the villi, there are clusters of large rod shaped bacteria.Sometimes necrosis is due to poor procedural execution and can be misinterpreted as lesions produced by bacteria. A telltale signs is the absence of bacteria in the histological section. Also, the blood vessels in the muscular layer are severely dilated by the accumulation of many degenerating erythrocytes indicative of vascular congestion.This was caused by ligated mesenteric blood vessels that likely were ligated to do excessive physical strain on the intestines. Ischemic loops can be easily identified microscopically during the surgical procedures. Following severe vascular congestion a few minutes after the ligature, the serosa will be dark blue instead of its normal pink reddish coloration.After watching this video, you should have a good understanding of how to create multiple intestinal loops in a chicken model by placing ligatures on the intestines. Once mastered the surgical procedures, excluding infection time, can be done in less than three hours, if it is performed properly. While attempting this procedure, it is important to remember to keep the chicken under deep anesthesia to control the pain and monitor vital signs during all the procedures, including surgical part and following infection time.Following this procedure or their methods like bacterial isolation, and straight identification such as gene detection by PCR can be performed in order to answer external questions like recovery and identification of the initial injected pathogen. After this development, this technique paved a way for researchers in the field of Clostridium perfringens pathogenesis to explore Necrotic Enteritis in chickens. Don't forget that working with live animals can be extremely variable.And precautions such as continuous anesthetic monitoring of the chicken should always be done while performing this procedure.

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Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...