*3.1. Establishment of Gut-Loop Model on a Cesarean-Born Neonatal Lamb*

In order to be able to compare the immunomodulatory properties of natural compounds in the intestinal response of newborn lambs without prior interference with natural microbial stimulation, we adapted the gut-loop model to cesarean-born lambs (Figure 1a). All physiological parameters were constantly recorded during the surgery (SpO2, ETCO2, respiratory and heart rates, body temperature) and no loss was registered. The ligatures were made in the ileum of lambs since it is the main infected area in the case of natural infection by *C. parvum*. We performed set of triplicates for each treatment to be used, separated by an interloop region to avoid diffusion of the treatment to another adjacent treatment to be administered (Figure 1b). Leakage between loops were never observed confirming the effective sealing of each loop. At the end of the loop's generation, an endto-end anastomosis was performed to reconnect the normal transit that will occur when lambs will first feed, and to allow the separation of the intestinal segment containing the isolated loops (Figure 1c). With the practice and experience gained over the first surgeries, the veterinary surgeon can now make around 25–30 intestinal loops (Figure 1d). The gut-loop lamb animals used to evaluate efficacy of immunostimulatory compounds were maintained with their littermates for 24 h before euthanasia and none presented abnormal behavior. Food intake was slightly lower in gut-loop lambs, but all animals were regularly fed on several occasions during the first 24 h (Figure 1e). In gut-loop animals, the body temperature drops before the beginning of the surgery and then regularly increases to reach similar temperature as littermate (Figure 1f), thanks to the heated neonatal resuscitation table, the heat lamp in the housing box and the early food intake following surgery.

## *3.2. Innate Immune Response to LPS Stimulation of Sterile Gut Loop*

Intestinal immune tolerance initiates within the first hours of life in response to microbial colonization gained during vaginal delivery and subsequently via colostrum, milk and multiple contact with environment. This immune tolerance is characterized by a rapid hypo responsiveness to microbial antigens as demonstrated in mouse models [12,13]. We aim to stimulate the immune responses of animals from birth with colostrum supplemented with natural products and in particular YCW fractions that contain TLR2 and TLR4 ligands. This led us to use lambs born by cesarean-section and whose intestine had not yet been in contact with a microbiota. In order to validate the requirement of using cesarean-born lambs, we assessed LPS responsiveness of isolated ileal loops and of the ileum exposed to microbial colonization 24 h after birth. Chemokines (CXCL1, CXCL2, CXCL8) and TNFα, known to be induced after LPS stimulation, were compared by transcriptomic analysis in both conditions. Our results (Figure 2) demonstrated that in isolated loops LPS induced a chemokine response and TNFα expression while in the connected ileum exposed to microbial and alimentary antigens, these levels were already upregulated and LPS stimulation was without clear additive effect except for a slight increase in cxcl8 mRNA expression. In order to compare the potential of microbial productsm it is therefore necessary to use a cesarean-born lamb that remains fully responsive to microbial ligands.

*Vet. Sci.* **2021**, *8*, x FOR PEER REVIEW 8 of 15

**Figure 2.** Intestinal responsiveness to LPS stimulation within the gut loop generated with a cesarean-born neonatal lamb. Intestinal tissues from the ileal gut loops and the ileum were recovered from the same neonatal lamb 24 h post-surgery. Small pieces of intestine, designed as "explants", were placed in a CO2 incubator at 37 °C in culture medium alone (untreated) or with LPS at a concentration of 10 µg/mL for 4 h (*n* = 3 explants per condition). Explants were processed for RNA extraction and *cxcl1* (**a**), *cxcl2* (**b**), *cxcl8* (**c**) and *tnfα* (**d**) gene expressions were quantified by RTqPCR. Results are expressed as 2e−ΔCt (mean ± SEM), following normalization with the expression of three reference genes (*hprt*; *gapdh*; *actb*). **Figure 2.** Intestinal responsiveness to LPS stimulation within the gut loop generated with a cesareanborn neonatal lamb. Intestinal tissues from the ileal gut loops and the ileum were recovered from the same neonatal lamb 24 h post-surgery. Small pieces of intestine, designed as "explants", were placed in a CO<sup>2</sup> incubator at 37 ◦C in culture medium alone (untreated) or with LPS at a concentration of 10 µg/mL for 4 h (*n* = 3 explants per condition). Explants were processed for RNA extraction and *cxcl1* (**a**), *cxcl2* (**b**), *cxcl8* (**c**) and *tnfα* (**d**) gene expressions were quantified by RT-qPCR. Results are expressed as 2e−∆Ct (mean <sup>±</sup> SEM), following normalization with the expression of three reference genes (*hprt*; *gapdh*; *actb*).

#### *3.3. Innate Ileal Immune Response to TLR-Ligands and YCW Fractions in Isolated Loops 3.3. Innate Ileal Immune Response to TLR-Ligands and YCW Fractions in Isolated Loops*

YCW principal components are β-glucans and mannoproteins, known to stimulate immune responses through multiple Pattern Recognition Receptors (PRR), mainly TLR2, TLR4 and Dectin-1 [18]. We investigated the innate immune response in ileal loops to two YCW from *S. cerevisiae*, differing in their polysaccharide composition. As controls, we used, *Escherichia coli* LPS, a TLR4-ligand, and R848, a synthetic TLR7-8 viral singlestranded ribonucleic acid mimic-ligand. We compared the responsiveness to these different agonists after 24 h of stimulation. YCW1 induced the mRNA expression of CXCL8 chemokine, IL1α, IL1β proinflammatory cytokines and the interferon-induced Mx1 while β-glucans enriched fraction only induced IL1β (Figure 3). LPS and R848 presented distinct cytokinic responses with R848 inducing the Mx1 upregulation while LPS was more prone to upregulate CXCL8 chemokine. These results confirm the potential of the neonatal gutloop model to be used as a screening method to evaluate the immunostimulating properties of various compounds using a single animal. YCW principal components are β-glucans and mannoproteins, known to stimulate immune responses through multiple Pattern Recognition Receptors (PRR), mainly TLR2, TLR4 and Dectin-1 [18]. We investigated the innate immune response in ileal loops to two YCW from *S. cerevisiae*, differing in their polysaccharide composition. As controls, we used, *Escherichia coli* LPS, a TLR4-ligand, and R848, a synthetic TLR7-8 viral single-stranded ribonucleic acid mimic-ligand. We compared the responsiveness to these different agonists after 24 h of stimulation. YCW1 induced the mRNA expression of CXCL8 chemokine, IL1α, IL1β proinflammatory cytokines and the interferon-induced Mx1 while β-glucans enriched fraction only induced IL1β (Figure 3). LPS and R848 presented distinct cytokinic responses with R848 inducing the Mx1 upregulation while LPS was more prone to upregulate CXCL8 chemokine. These results confirm the potential of the neonatal gut-loop model to be used as a screening method to evaluate the immunostimulating properties of various compounds using a single animal.

**Figure 3.** Innate ileal immune response to TLR-ligands and yeast cell wall fractions. Lamb's gutloops were stimulated in vivo immediately after the surgery procedure by in situ injection of LPS (10 µg/mL), R848 (10 µg/mL), yeast cell wall fraction 1 (YCW1) or 2 (YCW2) (5 mg/mL) into distinct intestinal loops. Twenty-four hours later, intestinal loops were recovered and samples processed for RNA extraction. *cxcl8* (**a**), *il1α* (**b**), *il1β* (**c**) and *mx1* (**d**) gene expressions were quantified by RT-qPCR. Results are expressed as 2e−ΔCt following normalization with the expression of three reference genes (*gapdh*; *hprt*; *actb*). **Figure 3.** Innate ileal immune response to TLR-ligands and yeast cell wall fractions. Lamb's gutloops were stimulated in vivo immediately after the surgery procedure by in situ injection of LPS (10 µg/mL), R848 (10 µg/mL), yeast cell wall fraction 1 (YCW1) or 2 (YCW2) (5 mg/mL) into distinct intestinal loops. Twenty-four hours later, intestinal loops were recovered and samples processed for RNA extraction. *cxcl8* (**a**), *il1α* (**b**), *il1β* (**c**) and *mx1* (**d**) gene expressions were quantified by RT-qPCR. Results are expressed as 2e−∆Ct following normalization with the expression of three reference genes (*gapdh*; *hprt*; *actb*).

#### *3.4. Evaluation of YCW Fractions on Early Cryptosporidium parvum Invasion and Development 3.4. Evaluation of YCW Fractions on Early Cryptosporidium parvum Invasion and Development*

To evaluate if immune stimulation with YCW fractions can reduce *C. parvum* infection with a limited number of animals, we used the benefit of the gut-loop model in providing multiple experimental conditions in a single animal. To evaluate *C. parvum* infection and development, we used a previously generated transgenic strain *Cp-Nluc*-INRAE allowing simple and accurate measurement of luciferase activity on tissue samples [16]. Two doses of *C. parvum* oocysts (1.5 × 104, 1.5 × 105) were inoculated in triplicate in independent loops and in both condition, parasite invasion and development were monitored by luciferase activity (Figure 4a). We confirmed the early *C. parvum* development within 24 h of infection by immunofluorescence microscopy, showing parasites developing into intestinal epithelial cells lining the ileal villi (Figure 4b). This result was also confirmed by RT-qPCR on *Cp18S* gene (Figure 4c) on tissue samples recovered from the same ileal loop as for luciferase activity. The correlation between the two variables presented a r2 of 0.26 and a *p* value of 0.0044 (Figure 4d). To evaluate if immune stimulation with YCW fractions can reduce *C. parvum* infection with a limited number of animals, we used the benefit of the gut-loop model in providing multiple experimental conditions in a single animal. To evaluate *C. parvum* infection and development, we used a previously generated transgenic strain *Cp-Nluc*-INRAE allowing simple and accurate measurement of luciferase activity on tissue samples [16]. Two doses of *C. parvum* oocysts (1.5 <sup>×</sup> <sup>10</sup><sup>4</sup> , 1.5 <sup>×</sup> <sup>10</sup><sup>5</sup> ) were inoculated in triplicate in independent loops and in both condition, parasite invasion and development were monitored by luciferase activity (Figure 4a). We confirmed the early *C. parvum* development within 24 h of infection by immunofluorescence microscopy, showing parasites developing into intestinal epithelial cells lining the ileal villi (Figure 4b). This result was also confirmed by RT-qPCR on *Cp18S* gene (Figure 4c) on tissue samples recovered from the same ileal loop as for luciferase activity. The correlation between the two variables presented a r<sup>2</sup> of 0.26 and a *p* value of 0.0044 (Figure 4d).

*Vet. Sci.* **2021**, *8*, x FOR PEER REVIEW 10 of 15

**Figure 4.** Establishment of *Cryptosporidium parvum* experimental infection in the gut-loop model. Data are cumulative results from three independent experimentations each performed with one newborn lamb. For each experimental condition, three loops were used; (**a**) Parasite load were determined by measuring luminescence activity in the ileal tissue from the loop 24 h after infection with oocysts of *C. parvum nluc*-INRAE transgenic strain (*n* = 9). Results are expressed as RLU per cm2 of intestinal tissue (mean ± SEM); (**b**) Immunofluorescence microscopy of ileal tissue section from a *C. parvum* infected gut loop (1.5 × 105 oocysts) 24 h after infection. Parasites were stained in red with anti-*C. parvum* antibodies and nuclei in blue with DAPI. "GC" corresponds to the germinal centers of the ileal Peyer's patch. White arrows indicate parasites developing into intestinal epithelial cells lining the ileal villi; (**c**) Quantification of *Cp18S* gene expression in the intestinal loops was performed on the same samples as above. RNA was extracted from a piece of each intestinal loop after 24 h of infection with *C. parvum nluc*-INRAE transgenic strain, reverse transcribed and amplified by quantitative PCR. Results are expressed as 2e−ΔCt (mean ± SEM) following normalization with the expression of three reference genes (*gapdh*; *hprt*; *actb*); (**d**) Correlation between parasite load evaluated by luminescence and *Cp18S* gene expression was determined by linear regression analysis. Statistical analyses in (**a**,**b**) were realized with the Kruskal–Wallis non-parametric test and the **Figure 4.** Establishment of *Cryptosporidium parvum* experimental infection in the gut-loop model. Data are cumulative results from three independent experimentations each performed with one newborn lamb. For each experimental condition, three loops were used; (**a**) Parasite load were determined by measuring luminescence activity in the ileal tissue from the loop 24 h after infection with oocysts of *C. parvum nluc*-INRAE transgenic strain (*n* = 9). Results are expressed as RLU per cm<sup>2</sup> of intestinal tissue (mean <sup>±</sup> SEM); (**b**) Immunofluorescence microscopy of ileal tissue section from a *C. parvum* infected gut loop (1.5 <sup>×</sup> <sup>10</sup><sup>5</sup> oocysts) 24 h after infection. Parasites were stained in red with anti-*C. parvum* antibodies and nuclei in blue with DAPI. "GC" corresponds to the germinal centers of the ileal Peyer's patch. White arrows indicate parasites developing into intestinal epithelial cells lining the ileal villi; (**c**) Quantification of *Cp18S* gene expression in the intestinal loops was performed on the same samples as above. RNA was extracted from a piece of each intestinal loop after 24 h of infection with *C. parvum nluc*-INRAE transgenic strain, reverse transcribed and amplified by quantitative PCR. Results are expressed as 2e−∆Ct (mean <sup>±</sup> SEM) following normalization with the expression of three reference genes (*gapdh*; *hprt*; *actb*); (**d**) Correlation between parasite load evaluated by luminescence and *Cp18S* gene expression was determined by linear regression analysis. Statistical analyses in (**a**,**b**) were realized with the Kruskal–Wallis non-parametric test and the Dunn's multiple comparison test, and significative difference was determined by a *p*-value < 0.05 (\*\* *p* < 0.01, \*\*\* *p* < 0.001).

Dunn's multiple comparison test, and significative difference was determined by a *p*-value < 0.05 (\*\* *p* < 0.01, \*\*\* *p* < 0.001). The control of cryptosporidiosis is still limited and the search of natural alternative highly anticipated. With the same animal and benefiting from the multiple experimental possible conditions offered by the gut-loop model, we investigated the ability of two YCW fractions to limit *C. parvum* development. Both yeast fractions presented a limited but significant effect on *C. parvum* development (Figure 5), thus validating the gut-loop model to The control of cryptosporidiosis is still limited and the search of natural alternative highly anticipated. With the same animal and benefiting from the multiple experimental possible conditions offered by the gut-loop model, we investigated the ability of two YCW fractions to limit *C. parvum* development. Both yeast fractions presented a limited but significant effect on *C. parvum* development (Figure 5), thus validating the gut-loop model to investigate new alternatives of treatment against this disease together with a limited use of experimental animals.

investigate new alternatives of treatment against this disease together with a limited use

of experimental animals.

**Figure 5.** Evaluation of yeast cell wall fractions on early *Cryptosporidium parvum* invasion and development. Data are cumulative results from three independent experimentations each with one newborn lamb. For each experimental condition, three loops were used. Loops were infected by in situ injection of 1.5 × 104 oocysts of *C. parvum nluc-INRAE* transgenic strain alone (untreated), or associated with YCW1 or YCW2 at a concentration of 5 mg/mL. Parasite loads were evaluated by quantifying luminescence in intestinal tissues after 24 h of infection. Results are expressed as RLU per cm2 of intestinal tissue with a logarithmic scale (mean ± SEM). Statistical analyses were performed with the Kruskal–Wallis non-parametric test followed by the Dunn's multiple comparison test (\*\* *p* < 0.01). **Figure 5.** Evaluation of yeast cell wall fractions on early *Cryptosporidium parvum* invasion and development. Data are cumulative results from three independent experimentations each with one newborn lamb. For each experimental condition, three loops were used. Loops were infected by in situ injection of 1.5 <sup>×</sup> <sup>10</sup><sup>4</sup> oocysts of *C. parvum nluc-INRAE* transgenic strain alone (untreated), or associated with YCW1 or YCW2 at a concentration of 5 mg/mL. Parasite loads were evaluated by quantifying luminescence in intestinal tissues after 24 h of infection. Results are expressed as RLU per cm<sup>2</sup> of intestinal tissue with a logarithmic scale (mean <sup>±</sup> SEM). Statistical analyses were performed with the Kruskal–Wallis non-parametric test followed by the Dunn's multiple comparison test (\*\* *p* < 0.01).

#### **4. Discussion 4. Discussion**

Neonates have an underdeveloped immune system at birth explaining their high susceptibility to infectious diseases. The high incidence of diarrhea in young ruminants must be addressed as soon as possible, as it results in the majority of mortality and morbidity [19,20]. Neonatal diseases, in particular those well controlled by neutralizing antibodies can often be prevented by maternal vaccination with vaccines administered before or during pregnancy. Multivalent vaccines against calf scours currently available are designed to prevent or limit infections with rotavirus, coronavirus, *E. coli* (K99) and *Salmonella* infections. There is no vaccine for *Cryptosporidium* to date which might be explained by the fact that antibodies seem to play a limited role in the protection process [21]. Therefore, alternative control strategies have to be investigated. Neonates have an underdeveloped immune system at birth explaining their high susceptibility to infectious diseases. The high incidence of diarrhea in young ruminants must be addressed as soon as possible, as it results in the majority of mortality and morbidity [19,20]. Neonatal diseases, in particular those well controlled by neutralizing antibodies can often be prevented by maternal vaccination with vaccines administered before or during pregnancy. Multivalent vaccines against calf scours currently available are designed to prevent or limit infections with rotavirus, coronavirus, *E. coli* (K99) and *Salmonella* infections. There is no vaccine for *Cryptosporidium* to date which might be explained by the fact that antibodies seem to play a limited role in the protection process [21]. Therefore, alternative control strategies have to be investigated.

To promote gut health and improve growth, supplements containing immunoglobulins or mineral and vitamin complements, or with probiotics such as *Enterococcus faecium* can be orally given to young ruminants just after birth [22–24]. Live yeast and yeast-derived products are also extensively used as probiotic and prebiotic, respectively, for farm animals to reduce the severity of diarrhea by preventing pathogenic bacteria from binding to intestinal epithelial cells or by modulating gut mucosal immunity [25–27]. Indeed, YCW fractions are known for their immunostimulatory properties, they harbor ligands for various innate receptor such as TLR2, TLR4 and Dectin-1. They have previously shown to be effective against cryptosporidiosis and pathogenic bacterial colonization in young ruminants. Mammeri et al. identified the anti-cryptosporidial activities of chitosan, a natural polysaccharide present in YCW in in vitro and mouse studies [28]. In another study, yeast culture enriched with mannan-oligosaccharides were fed in milk to Holstein heifer calves enrolled at 4 to 12 h of age, and this led to the presence of fewer *Escherichia coli* and pathogenic *E. coli* compared with control calves [29]. To promote gut health and improve growth, supplements containing immunoglobulins or mineral and vitamin complements, or with probiotics such as *Enterococcus faecium* can be orally given to young ruminants just after birth [22–24]. Live yeast and yeastderived products are also extensively used as probiotic and prebiotic, respectively, for farm animals to reduce the severity of diarrhea by preventing pathogenic bacteria from binding to intestinal epithelial cells or by modulating gut mucosal immunity [25–27]. Indeed, YCW fractions are known for their immunostimulatory properties, they harbor ligands for various innate receptor such as TLR2, TLR4 and Dectin-1. They have previously shown to be effective against cryptosporidiosis and pathogenic bacterial colonization in young ruminants. Mammeri et al. identified the anti-cryptosporidial activities of chitosan, a natural polysaccharide present in YCW in in vitro and mouse studies [28]. In another study, yeast culture enriched with mannan-oligosaccharides were fed in milk to Holstein heifer calves enrolled at 4 to 12 h of age, and this led to the presence of fewer *Escherichia coli* and pathogenic *E. coli* compared with control calves [29].

In addition to their immunostimulatory properties, YCW products are suggested to be anti-adhesive agents able to reduce adhesion of intestinal pathogens [30,31]. *Cryptosporidium spp.* are known to express the lectin galactose/*N*-acetyl-D-galactosamine, which facilitates their adhesion to host epithelial cells via interaction with sulfated proteoglycans [32,33]. Therefore, further investigations are required regarding the precise mechanism of In addition to their immunostimulatory properties, YCW products are suggested to be anti-adhesive agents able to reduce adhesion of intestinal pathogens [30,31]. *Cryptosporidium spp.* are known to express the lectin galactose/*N*-acetyl-D-galactosamine, which facilitates their adhesion to host epithelial cells via interaction with sulfated proteoglycans [32,33]. Therefore, further investigations are required regarding the precise

mechanism of inhibition of *C. parvum* observed with YCW extracts while considering the role of polysaccharide composition.

We also wish to investigate if immunostimulatory components administered with the colostrum can strengthen immune system of neonatal ruminants and reduce the incidence of cryptosporidiosis. Comparing multiple derivates from yeast or other sources will require the use of many animals. In order to follow reduce animal consumption, we aimed to develop a gut-loop model adapted to caesarean-born animals. This surgical model, although invasive by definition, fits well with the 3Rs principle. With the practice and experience gained over the first surgeries, we can now make around 25–30 intestinal loops within the ileal Peyer's patch area which gives the opportunity to produce replicates and/or evaluate many anti-infectious products with just a single newborn animal. With this gutloop model, the animal is its own control which reduces variability of responses especially in non-inbred animals that vary substantially in their genetic. We took a particular care to provide the best veterinary practices during surgery, anesthesia and analgesia, neonatal nursing, pre- and post-operative care. All of the contact related to the animals were optimized to avoid or limit pain and discomfort. After surgery, the animals fed naturally and did not show any particular behavior compared to its littermate.

The model is also flexible on the section of the intestine that could be investigated. Indeed, the small intestine contains distinct areas of organized lymphoid tissues at birth like Peyer's patches that are known to be the major inductive sites of immune responses. Ruminants possess jejunal Peyer's patches (JPP) that retain classical functions of intestinal Peyer's patches founds in mouse and human such as antigen sampling through Microfold cells and T- and B-cell activation, but also a peculiarly long ileal Peyer's Patch (IPP), which extends one meter along the terminal small intestine and which is known to be a primary lymphoid organ of B-cell development [34]. Since *C. parvum* infect primarily the distal small intestine, for our own investigations the intestinal loops were therefore generated in the ileum.

In order to compare various immunostimulant candidates containing TLR ligands we needed to use caesarean-section born lamb to have sterile intestinal environment mimicking the first encounter of the yeast derivates when administered with the first yeast-supplemented colostrum and putative interference with the presence of endotoxins that will raise rapidly in the gut lumen following microbiota installation. This process was previously reported in the mouse model by a mechanism that involves microRNA-146amediated translational repression and proteolytic degradation of the essential Toll-like receptor (TLR) signaling molecule interleukin-1 receptor-associated kinase 1 (IRAK1) [12]. This mechanism is sufficient to induce intestinal epithelial innate immune tolerance [13]. Although we did not demonstrate that similar mechanism occurs in lambs, we observed in this study that only in the gut loop explants, free of endotoxins, LPS induced chemokine production was preserved 5 h post-stimulation. Conversely, in explants generated with the ileum connected to the intestinal transit since 24 h, addition of LPS to these explants did not improve further chemokine upregulation with the exception of cxcl8 mRNA expression for which a slight increase was observed.

As a proof of concept, we next compared two YCW fractions for their immunostimulatory properties: one with a proportional content of β-glucans and mannoproteins and the second one enriched in β-glucans. We noticed that the YCW fractions enriched in β-glucans induced lower expression of pro-inflammatory cytokines in the gut-loop (CXCL8 and IL1α). Similar observation was previously made with macrophages cultured in vitro with *S. cerevisiae* extracts enriched in β-glucans that displayed weaker TLR2/4-related NFκB/AP-1 activity and less TNFα production [35]. We therefore can suspect that a similar mechanism may occur in gut lamb but this requires further investigations. When YCW1 and YCW2 were tested for their ability to reduce invasion and development of *C. parvum* in intestinal epithelial cells, they both limited in a modest but similar manner *C. parvum* early development despite different contents in β-glucans. One can conclude that the difference

in ability to induce higher level of these immune effectors did not play a significant role in the protection process.

In addition to the evaluation of products for their immunostimulatory or their antiinfectious properties, the gut-loop in cesarean-born lambs can be used to further investigate host–microbial interactions in a controlled environment, and decipher immune feature of a specific area of the intestine. The gut-loop system is indeed suitable for investigations in various intestinal lymphoid and non-lymphoid segments by just making loops in the selected area as performed with jejunal loop made in a one-month-old piglet to study innate immune response to *Salmonella* [36]. Since our model does not require the use of antibiotics after the loop surgery, the neonatal gut-loop model can also be very useful to investigate the role of selected microbiota or probiotics that could be introduced in the "sterile loops" to evaluate their capacity to promote intestinal immune responses and mucosae maturation. For the later, maintenance of gut loop for long period would be required. A model of fetal lamb with a single 8–10 cm intestinal loop model was previously generated, and in this case, sterile intestinal loop constructed in utero retained functional GALT for as long as 6–7 months after birth [14]. This therefore demonstrates that even longterm slow progressive enteric diseases can be investigated with the gut-loop model. Our lamb gut-loop model could also be a model for human cryptosporidiosis to evaluate drug compounds and innate immune responses, considering the observed similarities between those of young ruminants and young children infected by *Cryptosporidium*. However, it has some limitations; for example, the large ileal Peyer's patch of young ruminants, which is not present in human.

Overall, this model paves the way for further new control method development such as immunostimulants, antimicrobial compounds and vaccines dedicated to the control of enteric infections in neonates.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/vetsci8090170/s1, Table S1: List of forward and reverse primers used for gene expression analysis by RT-qPCR.

**Author Contributions:** Conceptualization, F.L., N.K.-H. and S.L.-L.; methodology, A.B., C.B., F.L., J.C., N.K.-H. and S.L.-L.; formal analysis, A.B., F.L., N.K.-H. and S.L.-L.; investigation, A.B., A.P., C.B., J.C., J.P., N.K.-H. and S.L.-L.; writing—original draft preparation, A.B., F.L., J.C., J.S., N.K.-H., P.P.-P. and S.L.-L.; funding acquisition, F.L., J.S., P.P.-P. and S.L.-L. All authors have read and agreed to the published version of the manuscript.

**Funding:** A.B. benefited from a PhD grant from a CIFRE fellowship (Industrial Research Training Agreement) with Phileo by Lesaffre. This research was funded by both INRAE and PHILEO BY LESAFFRE funds.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Val de Loire (CEEA VdL n◦19) (protocol code: APAFlS#16870-201809261558973 v2 and date of approval: 14 February 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are openly available in Zenodo at [10.5281/zenodo.5235377].

**Acknowledgments:** We would like to thank Léa Bouyonnet for generating the figure on the intestinalloops with Illustrator® (Adobe, San José, CA, USA) and Eric Auclair and Christine Julien (Phileo by Lesaffre, Marcq-en-Baroeul, France) for their constructive discussions. We are also very grateful to Caroline Thérésine, Tiffany Pezier (ISP UMR, INRAE) and Fanny Faurie-Sarce (PFIE, INRAE) for the production of the *C. parvum nluc*-INRAE transgenic strain and to Aude Remot and Pierre Germon (ISP UMR, INRAE) for providing some primer sequences.

**Conflicts of Interest:** The authors declared no potential conflict of interest with respect to the research, authorship, and/or publication of this article.
