**1. Introduction**

Infectious diseases are mainly controlled by the use of vaccines and antimicrobials. Despite major discoveries, some infectious diseases remained refractory to vaccination and treatments and therefore require specific attention together with the new infectious zoonotic threats that increasingly emerge these last decades. The evaluation of new antimicrobials is usually first performed by bioinformatics and/or by in vitro approaches sometimes resulting from High-Throughput Screening (HTS) on miniaturized 2D cell-based assays, evaluating 100,000 or more samples per day [1,2]. Many of these candidates failed when entering clinical trials and have required the unnecessary use of numerous experimental animals. Ex vivo cultures of multipotent or pluripotent stem cells in a three-dimensional (3D) matrix represent major improvements and contribute to reducing animal use [3,4]. However, despite their obvious advantages, these systems cannot yet fully reproduce all the complex characteristics and interactions encountered in vivo, such as the (I) multiplicity and spatial organization of cell types, (II) the immune cell recruitments, (III) the ability to

**Citation:** Baillou, A.; Kasal-Hoc, N.; Barc, C.; Cognié, J.; Pinard, A.; Pezant, J.; Schulthess, J.; Peltier-Pain, P.; Lacroix-Lamandé, S.; Laurent, F. Establishment of a Newborn Lamb Gut-Loop Model to Evaluate New Methods of Enteric Disease Control and Reduce Experimental Animal Use. *Vet. Sci.* **2021**, *8*, 170. https:// doi.org/10.3390/vetsci8090170

Academic Editors: Ana Faustino and Paula A. Oliveira

Received: 30 July 2021 Accepted: 20 August 2021 Published: 24 August 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

experiment in presence of a full cultivable and non-cultivable microbiota or (IV) to reproduce the peculiarity of a specific age of development e.g., neonatal period, etc. In addition, these models are usually unsuitable for assessing the mechanism of physiopathology resulting from infectious processes. Until technical progress can resolve these limitations, it is therefore important to develop or improve in vivo methods to minimize the use of animals and take full advantage of each experiment in accordance with the 3R rules (Replacement, Reduction and Refinement) [5].

Neonatal enteric infections result essentially from the infection of bacterial, parasitic, or viral pathogens and they have profound effects on intestinal absorption, nutrition, and youth development as well as on global mortality. Cryptosporidiosis due to the zoonotic protozoan parasite *Cryptosporidium parvum* (*C. parvum*) is characterized by infection of the epithelial cells of the small intestine, mainly in the ileum, leading to acute diarrhea and dehydration that may lead to death in severe cases [6]. This disease represents a true one-health threat with severe consequences for human and animal health [7,8]. Extensive new epidemiology studies revealed cryptosporidiosis to be the second leading cause of death in children due to diarrheal disease worldwide [9], and is the first cause of diarrheal enteric disease in young ruminants in France [10]. There is no vaccine and a very limited chemotherapy available for animals and humans [11].

We aim to evaluate natural alternatives for controlling cryptosporidiosis using lambs as a target species and also as a model for larger animals such as calves. Indeed, lambs represent a cost-effective model and harbor similar development of gut lymphoid tissues at birth to other young ruminants. We intent to stimulate the immune responses of animals from birth with colostrum supplemented with natural products such as yeast cell wall (YCW) fractions that contain TLR2, TLR4 and Dectin-1 receptor ligands. However, intestinal immune tolerance initiates rapidly after birth in response to microbial colonization gained during vaginal delivery and subsequently via colostrum, milk and multiple contacts with the environment. This immune tolerance is characterized by a rapid hypo-responsiveness to microbial antigens as demonstrated in mouse models [12,13].

We therefore developed an in vivo model suitable to investigate both host–pathogen interactions in a controlled environment and to evaluate new natural antiparasitic compounds. Our model relies on two previously described gut-loop models: one performed with fetal lambs (120 to 130 days of gestation) but with just a single loop [14] and another one made with 4–6 month-old lambs [15]. The surgical procedure was therefore successfully adapted right after birth to cesarean-born lambs and allowed to produce an isolated intestinal segment in the ileum area free of microbiota and immune system stimulation. Multiple loops were created in the isolated intestinal segment which allowed for the evaluation endotoxin responsiveness, immune responses, parasite replication in presence or not of YCW fractions. This new model combines different specificities which were required for our investigations such as (1) a newborn model for neonatal enteric disease study, (2) sterility for evaluating the immunomodulatory properties of natural compounds provided in the first colostrum and (3) a large number of intestinal loops per animal to perform multiple comparisons of selected compounds while severely limiting the use of experimental animals.

#### **2. Materials and Methods**

#### *2.1. Development of a New Animal Model*

#### 2.1.1. Ethic Statements

Animal needs were met in accordance with the European Community Council Directive 2010/63/EU (Decree: 2013-118 01/02/2013). The experimental facilities had received authorization to house experimental animals from the local bureau of veterinary services (Indre-et-Loire, France, authorization N◦ : D 37-175-3), and all the experimental procedures were approved by the Val de Loire Ethics Committee (authorization N◦ APAFlS#16870- 201809261558973 v2). All animal experimentations have been performed in the Infectiology of Farm, Model and Wildlife Animals Facility (PFIE, Centre INRAE Val de Loire available

online: https://doi.org/10.15454/1.5572352821559333E12 (accessed on 23 August 2021); member of the National Infrastructure EMERG'IN). All the personnel involved had special training in animal care, handling and experimentation, as required by the French Ministry of Agriculture.

#### 2.1.2. Neonatal Care of Newborn Lambs after Cesarean Surgery

The pregnant Préalpes-du-Sud ewe selected to give birth to two lambs was subjected to classical cesarean surgery. After stimulation of the respiratory function, right after birth, they were positioned in lateral decubitus and received intranasally one to two drops of doxapram (Dopram®, Vetoquinol, Lure, France) to stimulate the respiratory rate. The umbilical cord was disinfected with Vetedine® (Vetoquinol, Lure, France) and newborns were then placed together in close contact in the housing box, in a dry and warm environment, under a heating lamp. After 30 min, one of the two lambs was operated.

#### 2.1.3. Intestinal Loop Surgery

Preparation: The lamb was placed on a heated neonatal resuscitation table and received isoflurane via a face mask for general anesthesia and buprenorphine (Buprecare® Multidose 0.01 mg/kg, Axience, Pantin, France) for analgesia by intramuscular injection. The lamb was then intubated with an endotracheal tube (internal diameter 4 to 5 mm, RÜSCH®, Teleflex, Wayne, NJ, USA) and put on assisted ventilation (CPV mode: controlled pressure ventilation, Fabius® Tiro, Dräger, Lübeck, Germany), with a 50/50 oxygen and air mixture. The monitoring system installed (BLT, M8500) allowed to continuously monitor rectal temperature, heart and respiratory rates, oxygen saturation (SpO2) and End Tidal CO<sup>2</sup> (ETCO2). An intravenous catheter was placed in the cephalic vein and a warm infusion of sterile isotonic Ringer-Lactate saline solution (Osalia, Paris, France) was started at a rate of around 10 mL/kg/h via a flow regulator.

Surgical intervention: The surgical area was disinfected and a subcutaneous injection of 1 mL of lidocaine was performed at the level of the midline. After a few minutes, a 10-cm skin incision was made in the midline, caudal to the umbilicus. After dissection of the subcutaneous tissue and opening of the abdominal cavity, the urachus duct was reclined. Sterile compresses moistened with prewarmed (+37 ◦C) sterile saline were placed all around the surgical wound. The intestinal tract was exteriorized on the compresses, the area of interest (from the ileocecal fold) was identified and the rest was reintroduced into the abdomen. The externalized portion was regularly humidified with sterile warm saline throughout the procedure.

Creation of loops and inter-loops: A silk ligature (dec. 3, SILK®, SMI, St Vith, Belgium) was made approximately at 5 cm from the ileocecal fold (retrograde direction). This 5 cm area is the site of the distal enterotomy. Then, moving up the small intestine, ligatures were placed at approximately 2–3 cm intervals to generate three consecutive loops, and an interloop region which separates each set of triplicates. This can be repeated as many times as necessary before the continuous ileal Peyer's patch disappears (Figure 1b). Long ends were performed to allow the identification of each triplicate of loops plus the interloop region to be easily clamped for reliable tracking during the loop injection stage. The lumen of the gastrointestinal tract was not rinsed before the ligatures were made, the intestinal contents remained therefore intact.

**Figure 1.** Intestinal loops surgery on the ileum of cesarean-section born lamb. (**a**) Picture of lamb that was anesthetized before surgery, intubated with endotracheal tube and put on assisted ventilation; (**b**) Schematic representation of the intestinal loop generation in the ileum of caesarean-born lamb. Series of triplicates of loops (L1, L2, L3) were generated between the two areas (I and II) indicated by arrows. Interloop region separates series of triplicates and are indicated in shaded areas. The first ligature of the triplicates harbors longer ends, so do the one of each triplicate to facilitate the identification; (**c**) Schematic representation of end-to-end anastomosis is visualized (I/II) allowing intestinal transit restauration and the separation of the intestinal segment containing the isolated loops; (**d**) Picture of a loop creation within intestinal segment during surgery; (**e**) Food intake represented by the volume of milk drunk per ml and per kg of lamb body weight per 24 h for the gut-loop lamb and its littermate. Each point shape corresponds to an independent experimentation (*n* = 4 for each group); (**f**) Body temperature (°C) (mean ± SEM) was monitored from birth to 20-h post-surgery (ps) for the gutloop lamb and its littermate. The grey box corresponds to the surgery intervention (from start to end). **Figure 1.** Intestinal loops surgery on the ileum of cesarean-section born lamb. (**a**) Picture of lamb that was anesthetized before surgery, intubated with endotracheal tube and put on assisted ventilation; (**b**) Schematic representation of the intestinal loop generation in the ileum of caesarean-born lamb. Series of triplicates of loops (L1, L2, L3) were generated between the two areas (I and II) indicated by arrows. Interloop region separates series of triplicates and are indicated in shaded areas. The first ligature of the triplicates harbors longer ends, so do the one of each triplicate to facilitate the identification; (**c**) Schematic representation of end-to-end anastomosis is visualized (I/II) allowing intestinal transit restauration and the separation of the intestinal segment containing the isolated loops; (**d**) Picture of a loop creation within intestinal segment during surgery; (**e**) Food intake represented by the volume of milk drunk per ml and per kg of lamb body weight per 24 h for the gut-loop lamb and its littermate. Each point shape corresponds to an independent experimentation (*n* = 4 for each group); (**f**) Body temperature (◦C) (mean ± SEM) was monitored from birth to 20-h post-surgery (ps) for the gut-loop lamb and its littermate. The grey box corresponds to the surgery intervention (from start to end).

> Double enterotomy, end-to-end anastomosis: A double enterotomy was performed on each extremity of the area containing loops (Figure 1c). The four ends created were immediately wiped with a sterile compress moistened with warm saline. To avoid leakage or adhesion, each end of the isolated intestinal segment was sutured with a single suture (dec. 1.5, MONOCRYL®, Johnson & Johnson, New Brunswick, NJ, USA) and a jejuno–ileal anastomosis was performed to allow normal intestinal transit before gently placing the entire externalized digestive tract back into the abdominal cavity. It should be noted that the vascularization of the intestinal loops after the separation of the ileal segment was maintained. The abdominal cavity was rinsed several times with sterile warm saline. The abdominal wall was stitched with a single suture (dec. 2, SAFIL®, B. BRAUN, Melsungen, Germany) followed by subcutaneous and cutaneous sutures and finally, the wound was disinfected. Isoflurane supply was cut off, the animal was put on assisted ventilation with Double enterotomy, end-to-end anastomosis: A double enterotomy was performed on each extremity of the area containing loops (Figure 1c). The four ends created were immediately wiped with a sterile compress moistened with warm saline. To avoid leakage or adhesion, each end of the isolated intestinal segment was sutured with a single suture (dec. 1.5, MONOCRYL®, Johnson & Johnson, New Brunswick, NJ, USA) and a jejuno–ileal anastomosis was performed to allow normal intestinal transit before gently placing the entire externalized digestive tract back into the abdominal cavity. It should be noted that the vascularization of the intestinal loops after the separation of the ileal segment was maintained. The abdominal cavity was rinsed several times with sterile warm saline. The abdominal wall was stitched with a single suture (dec. 2, SAFIL®, B. BRAUN, Melsungen, Germany) followed by subcutaneous and cutaneous sutures and finally, the wound was disinfected. Isoflurane supply was cut off, the animal was put on assisted ventilation

> a 100% oxygen mixture and then positioned in a lateral recumbence to accelerate awak-

with a 100% oxygen mixture and then positioned in a lateral recumbence to accelerate awakening. The animal was regularly "switched" to spontaneous ventilation (Man/Spont) and received an intravenous injection of doxapram (0.87 mg/kg, Dopram-V Injectable®, Vetoquinol, Lure, France), repeated once if necessary, to help spontaneous breathing. The animal was then extubated, the catheter was removed and the animal was placed in the housing box with heat lamp, in direct contact with the control lamb from the litter.

Post-operative care: The lambs were fed as the first signs of awakening were observed (Colostromix®, Technovet Eurotonic, Briec, France). The volume and number of feedings were adapted upon lamb request. Analgesia was continued with four intramuscular injections per day of buprenorphine (0.01 mg/kg). The criteria for assessing the animal's clinical condition and possible pain were primarily behavioral: vocalizations (which do not occur after drinking), lordosis, prostration or apathy, absence or too frequent stands up, disinterest in the surroundings, low head carriage or low ears, etc. At every visit to feed them (3 h interval), the surgical wound, rectal temperature, quantity of ingested milk, stool and urine output were controlled. No additional enrichment was provided other than the presence of a littermate from the same litter and all physical contact with the animal technicians, particularly during feedings, which is particularly important at this physiological stage.

Sample recovery: After 24 h, the animal was injected intramuscularly with xylazine (0.5 mg/kg, ROMPUN®, Bayer, Leverkusen, Germany) and euthanized intravenously with pentobarbital (180 mg/kg, DOLETHAL®, Vetoquinol, Lure, France). The animal was then bled and the small and large intestine was harvested in a whole (duodenum to colon), then samples were taken from each intestinal loop. The unique 24 h-time point was selected to limit animal consumption based on the compromise between assessing the innate immune response to immunostimulants and allowing sufficient time to measure the effect on parasite invasion and development.
