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

Summary

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

Abstract

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.

Introduction

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 condition^2,3,4,5. 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 'loops' 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 candidates^10,11. 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 mice^2,9, 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's skill and experience, as accurate procedures are essential for the success of the project.

Protocol

All procedures with live animal use were authorized by the Ethical Committee of the Faculty of Veterinary Medicine, Université de Montréal (CÉUA, 'Comité d'éthique de l'utilisation des Animaux').

1. Considerations Before Surgery

1. 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.
2. Withdraw the feed 12 h prior to the procedures to empty the intestinal tract.
3. Prior to surgery, place the chickens under general anesthesia following a protocol verified by an anesthesiologist. Premedicate each chicken 15 min prior to the surgery with an intramuscular (IM) injection of midazolam 1 mg/kg and butorphanol 4 mg/kg. Repeat, every 4 h, the injection of butorphanol 4 mg/kg IM or when the breathing pattern changes and frequency increases during anesthesia. For the general anesthesia, during all procedures, administer isoflurane at a concentration between 1.5 and 2.5% with an intratracheal tube.
   NOTE: The use of premedication for sedation and analgesia is recommended as they will significantly improve the depth of anesthesia, reduce the quantity of anesthetics necessary during all procedures, as well as enable researchers to control pain levels.
4. Monitor the anesthesia by recording body temperature, heart rate and electrocardiogram (ECG), respiratory rate and rhythm, oxygen saturation, expired carbon dioxide (CO₂) and neurologic reflexes.
   NOTE: A person must be assigned to monitor these parameters throughout all the procedures.
5. Follow sterility principles throughout the surgical procedures. Ensure that the surgeon and the assistant surgeon dress with sterile gowns, gloves, a mask and protective goggles according to institutional preoperative protocols. Ensure that the surgeon scrubs the hands with a chlorhexidine gluconate impregnated sterile brush up to the elbow with emphasis on fingernails and each finger surface.
6. Sterilize the surgical instruments with a sterilizer.

2. Surgical Site Preparation

1. Firstly, manually remove the feathers from the abdomen with a gentle traction.
2. Scrub the surgical site with a chlorhexidine gluconate detergent impregnated sterile brush. Gently scrub the skin from the center to the outside for a total contact time of 5 min without coming back to the center.
3. Perform 3 alternative passages on the surgical site with a chlorhexidine gluconate solution and isopropyl alcohol using sterile gauzes.
   NOTE: The passage must start from the middle of the surgical site to the borders to avoid contamination from the non-disinfected areas to the surgical site.
4. Place the sterile surgical drape on the chicken.

3. Extraction of the Small Intestines from the Abdominal Cavity

1. Incise the surgical drape with scissors to expose only the skin of the surgical site.
2. Incise the skin by performing an ''L'' shape low-midline incision with a scalpel blade #3.
   1. Start the first incision 1 cm caudal to the sternum and end it at 1 cm cranial to the cloaca.
   2. Perform the second incision perpendicular to the first incision. Start from the caudal end of the first incision and continue 5 cm to the left side of the abdomen by following the pelvis line.
      NOTE: This opening will create a flap that will allow an easier extraction of the intestines.
3. With surgery scissors, cut the peritoneum and abdominal muscles with the same ''L'' shape pattern to open the abdominal cavity.
   NOTE: This will expose the air sacs. Intestines are located underneath these structures. Air sacs must be humidified with gauzes saturated with sterile saline 0.85 % to avoid desiccation and possible rupture throughout the procedure.
4. Insert a Snook spay hook in the abdominal cavity by following the left abdominal wall and work the way around the abdominal air sacs.
5. Gently exteriorize the intestines and spread these out on the surgical field.
   NOTE: Keep intestines humidified by frequently spraying sterile saline 0.9% throughout the procedure. Also, gauzes humidified with sterile saline can be placed on the intestines to avoid desiccation.
6. Grab the intestines using a dry sterile gauze and gently pull out the small intestines by manual traction to expose the jejunum.
   NOTE: Mesenteric vessels are fragile and excessive tension may rupture them which may lead to intestinal ischemic lesions and greatly compromise the outcome of the procedure.

4. Fabrication of Intestinal Loops

1. Creation of loop no. 1.
   1. Place a simple ligature with a polyglactin multifilament synthetic absorbable material in the proximal jejunum by avoiding the ligature of major mesenteric vessels.
   2. Place a distal ligature 2 cm away from the proximal ligature.
      NOTE: Simple ligatures made with multifilament suture material are placed along the intestinal tract to create hermetic segments. A schema (Figure 1) describes the location of all ligatures. The loops consist of a proximal and distal ligature separated by 2 cm.
2. Creation of interloop no. 1.
   1. Place a simple ligature 0.5 cm aborally to the distal ligature of loop no.1.
      NOTE: Between successive loops, there are interloops of 1 cm, i.e. segments making a physical separation between the loops. To likely decrease the possible risk of cross-contamination from one loop to another, a simple ligature is placed in the middle of the interloop (0.5 cm from the distal and proximal ligature of 2 adjacent loops). This reduces the risk of a possible leaked contaminant from a loop to reach the ligature of an adjacent loop. These segments are not mandatory but will decrease the risk of cross-contamination.
3. Creation of subsequent loops and interloops.
   1. Repeat steps 4.1 and 4.2 to obtain the number of desired loops.
      NOTE: In this experiment, 9 loops and 8 interloops were created. Total length amounted to 26 cm. The number of loops can be adjusted depending on the study.

5. Injection of Clostridium perfringens Strains Into the Loops

1. Inject with a sterile syringe and needle 26 G the pathogen in loop no.1. Gently insert the needle on the antimesenteric side of the intestine at a 45° angle.
   NOTE: In this experiment, 0.2 mL of brain heart infusion (BHI) containing 1 x 10⁶ CFU (colony forming units) of C. perfringens in the mid-log growth phase was injected in each loop. 5 different strains were injected in 5 different loops and the 4 remaining loops were injected with a negative control containing no bacteria (sterile BHI), alternating pathogens and sterile BHI loops.
2. Repeat step 5.1 to inject all loops.
3. Gently replace the intestinal tract in the dorsal area of the abdominal cavity, underneath the air sacs.

6. Closure of the Abdominal Cavity

1. The suture of the peritoneum and abdominal muscles.
   1. Suture the peritoneum and abdominal muscles along the pelvis (made in step 3.3) with a simple continuous suture pattern using a polyglactin multifilament synthetic absorbable material.
   2. Suture the peritoneum and abdominal muscles from the sternum to the cloaca (made in step 3.3. with a simple continuous suture pattern using a polyglactin multifilament synthetic absorbable material.
2. The suture of the skin.
   1. Using a polyglactin multifilament synthetic material and a simple continuous suture pattern, close the incision of the skin along the pelvis made in step 3.2.
   2. Using the same multifilament ligature material and a simple continuous suture pattern, close the incision of the skin from the sternum to the cloaca made in step 3.2.

7. Conclusion

1. After the surgical procedures, 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.
2. After the desired infection time, humanely euthanize the chicken by cervical dislocation under general anesthesia.
   NOTE: For these procedures, the required time to induce microscopic lesions with C. perfringens pathogenic strains was 7 h.
3. With a scalpel blade #3, cut a 0.5 cm to 1 cm long loop section and place it in 10% formalin for fixation overnight and further histopathological analysis.
4. With the same scalpel blade, cut the remaining loop section and place it in a sterile microcentrifuge tube for the isolation of the C. perfringens strains in each loop further bacteriologic analysis for their genetic characterization.
5. Repeat steps 7.2 and 7.3 for all loops by changing the scalpel blade between every loop.

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...

Discussion

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 enterica^7,9,13,14,15. It has also been used to analyze the mucosal immune response

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EZ-Scrub 747	Becton Dickinson and Company		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
Isopropyl alcohol 70% USP 4 L	Commercial isopropyl alcohol	P016IP70	Web address: http://www.comalc.com/products/
Chlorexidine	Sigma-Aldrich	282227-1G	Chlorhexidine gluconate solution, must be diluted to 4%. Web address: http://www.sigmaaldrich.com/catalog/product/aldrich/282227?lang=fr®ion=CA&gclid=EAIaIQ obChMI8vHr4_OY1wIV3rrACh2Z WQKuEAAYAiAAEgLKx_D_BwE
Surgical Drape Small (27 inch x 24 inch) with 3 x 6 inch Fenestration	Veterinary Specialty Products	32724	Web address: http://www.vetspecialtyproducts.com/index.cfm?fuseaction=ecommercecatalog. detail&productgroup_id=15
Scalpel blades #3	Swann-Morton	301	Web address: https://www.swann-morton.com/product/16.php
Sterile saline NaCl 0.9%	Sigma-Aldrich	S8776-100ML	Web address: http://www.sigmaaldrich.com/catalog/product/sigma/s8776?lang=fr®ion=CA
Vicryl 3-0	Ethicon	D9003	Polyglactin multifilament absorbable suture material. Web address: http://www.ethicon.com/healthcare-professionals/products/wound-closure/absorbable-sutures/coated-vicryl-polyglactin-910-suture
Syringe 1 ml with 26G needle	Becton Dickinson and Company	329652	Web address: https://www.bd.com/en-us/offerings/capabilities/diabetes-care/insulin-syringes/bd-1-ml-conventional-insulin-syringes
Brain Heart Infusion	Sigma-Aldrich	1104930500	Web address: http://www.sigmaaldrich.com/catalog/product/mm/110493?lang=fr®ion=CA&cm_sp=Insite-_-prodRecCold_xorders-_-prodRecCold2-1
Eppendorf tube	Sigma-Aldrich	T9661-500EA	Microcentrifuge tube. Web address: http://www.sigmaaldrich.com/catalog/product/sigma/t9661?lang=fr®ion=CA&gclid=EAIaIQ obChMI-YizzfiY1wIVRkCGCh 30SQjuEAAYAiAAEgLK5fD_BwE
Formalin solution, buffered neutral, 10%	Sigma-Aldrich	HT501128-4L	Ratio tissue : formalin of 1: 10 for adequate fixation. Web address: http://www.sigmaaldrich.com/catalog/product/sigma/ht501128?lang=fr®ion=CA
Clostridium perfringens strains	Université de Montréal, Chaire en recherche avicole	N/A	Specific to each laboratory, available upon request to correspondant author