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, i....

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....

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