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

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.