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Immunohistochemical analysis of bovine small intestine: (A) haematoxylin and eosin histological staining to identify distinct crypt and villus structures from four different locations (#1, #2, #3, and #4) in the jejunum between the duodenum and ileum. Scale bar: 300 μm; (B) the number of intestinal organoids per basement matrix dome to verify the efficiency of the derivation of intestinal organoids from four different locations. The number of intestinal organoids derived from location #1 in the jejunum close to the duodenum was significantly higher, compared to locations #3 and #4. The values are the means plus the standard error of mean (S.E.M) and different letters (a-d) indicate significant differences (p < 0.05); (C) immunohistochemistry of LGR5, Bmi1, F-actin, and Chromogranin A in bovine small intestine. The fluorescently stained crypts were counterstained with diamidino-2-phenylindole (DAPI). Scale bar: 20 μm.
Source publication
Intestinal organoids offer great promise for disease-modelling-based host–pathogen interactions and nutritional research for feed efficiency measurement in livestock and regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterisation and three-dimensional (3D) expansion of adult stem ce...
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... intestinal tissue sections were subjected to anatomical analysis using haematoxylin and eosin histological staining to identify distinct crypt and villus structures. As shown in Figure 2A, locations #1 and #2 were better developed than locations #3 and #4. The detailed view from vertical and horizontal sections in locations #1 and #2 showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side (Supplementary Figure S2). ...
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... shown in Figure 2A, locations #1 and #2 were better developed than locations #3 and #4. The detailed view from vertical and horizontal sections in locations #1 and #2 showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side (Supplementary Figure S2). Furthermore, to verify the efficiency of the derivation of intestinal organoids from four different locations, we subsequently cultivated intestinal organoids. ...
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... to verify the efficiency of the derivation of intestinal organoids from four different locations, we subsequently cultivated intestinal organoids. Based on the results, the number of intestinal organoids per basement matrix dome was highest in location #1 ( Figure 2B), indicating the most growth potential for the derivation of intestinal organoids. In addition, to identify intestinal stem cells in vivo, immunohistochemistry with respect to several markers involved in intestinal stem cells and epithelial cells was conducted. ...
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... addition, to identify intestinal stem cells in vivo, immunohistochemistry with respect to several markers involved in intestinal stem cells and epithelial cells was conducted. As shown in Figure 2C, intestinal crypts isolated from location #1 of the small intestine had a distinct expression, such as leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a key gene required for stemness that is expressed in columnar crypt cells, B lymphoma Mo-MLV insertion region 1 homology (Bmi1), which was found in +4 cells adjacent to Paneth cells and F-actin in the intestinal epithelial cytoskeleton. Moreover, the fluorescently stained crypts showed epithelium-specific expression of Mucin2 in goblet cells, Ecadherin in adherent junctions (Supplementary Figure S3). ...
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... intestinal tissue sections were subjected to anatomical analysis using haematoxylin and eosin histological staining to identify distinct crypt and villus structures. As shown in Figure 2A, locations #1 and #2 were better developed than locations #3 and #4. The detailed view from vertical and horizontal sections in locations #1 and #2 showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side (Supplementary Figure S2). ...
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... shown in Figure 2A, locations #1 and #2 were better developed than locations #3 and #4. The detailed view from vertical and horizontal sections in locations #1 and #2 showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side (Supplementary Figure S2). Furthermore, to verify the efficiency of the derivation of intestinal organoids from four different locations, we subsequently cultivated intestinal organoids. ...
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... to verify the efficiency of the derivation of intestinal organoids from four different locations, we subsequently cultivated intestinal organoids. Based on the results, the number of intestinal organoids per basement matrix dome was highest in location #1 (Figure 2B), indicating the most growth potential for the derivation of intestinal organoids. In addition, to identify intestinal stem cells in vivo, immunohistochemistry with respect to several markers involved in intestinal stem cells and epithelial cells was conducted. ...
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... addition, to identify intestinal stem cells in vivo, immunohistochemistry with respect to several markers involved in intestinal stem cells and epithelial cells was conducted. As shown in Figure 2C, intestinal crypts isolated from location #1 of the small intestine had a distinct expression, such as leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a key gene required for stemness that is expressed in columnar crypt cells, B lymphoma Mo-MLV insertion region 1 homology (Bmi1), which was found in +4 cells adjacent to Paneth cells and F-actin in the intestinal epithelial cytoskeleton. Moreover, the fluorescently stained crypts showed epithelium-specific expression of Mucin2 in goblet cells, E-cadherin in adherent junctions (Supplementary Figure S3). ...
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... addition, we investigated the paracellular permeability character of the epithelial layer using fluorescent tracers up to 4 hr after treatment. FITC-dextran labelled the organoid lumen, demonstrating a high permeability for compounds of up to 4 kDa, such as glucose, peptides, and Figure 2. Immunohistochemical analysis of bovine small intestine: (A) haematoxylin and eosin histological staining to identify distinct crypt and villus structures from four different locations (#1, #2, #3, and #4) in the jejunum between the duodenum and ileum. ...
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... to assess the position effects for efficient isolation and derivation of intestinal organoids along the length of the small intestine in bovine, we sectioned four different locations in the jejunum between the duodenum and ileum. Interestingly, we found that jejunum (location #1 and #2) close to the duodenum showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side and intestinal crypts isolated from location #1 in the jejunum close to the duodenum had the most growth potential for the derivation of intestinal organoids (Figure 2). However, mouse models have shown that more distal tissues in the foetal intestine can be formed organoids well [37], suggesting the regional differences in vitro growth potential. ...
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... organoids show various developmental stages and can be spheroidal (round shaped), show budding (spheroids with extension), and have mature villus and crypt-like structures (branched structures). The detailed structures represent organoid propagation from day 2 to the fully grown structure on day 8. Scale bar: 100 µm (day 0, day 2) and 50 µm (day 3, day 6, and day 8), Figure S2: Histological H and E staining of the vertical and horizontal views reveal the positions of the intestinal epithelial glands, such as crypts (oval structures at the bottom) and villi (finger-like structures) cells, with varying magnification (40×, 100×, and 200×) at four different locations from #1 to #4. The purple spots show nuclei and the epithelium is stained pink, Figure S3: immunohistochemistry of Bmi1, E-cadherin, Chromogranin A, and Muccin2 in bovine small intestine. ...
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... intestinal tissue sections were subjected to anatomical analysis using haematoxylin and eosin histological staining to identify distinct crypt and villus structures. As shown in Figure 2A, locations #1 and #2 were better developed than locations #3 and #4. The detailed view from vertical and horizontal sections in locations #1 and #2 showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side (Supplementary Figure S2). ...
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... shown in Figure 2A, locations #1 and #2 were better developed than locations #3 and #4. The detailed view from vertical and horizontal sections in locations #1 and #2 showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side (Supplementary Figure S2). Furthermore, to verify the efficiency of the derivation of intestinal organoids from four different locations, we subsequently cultivated intestinal organoids. ...
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... to verify the efficiency of the derivation of intestinal organoids from four different locations, we subsequently cultivated intestinal organoids. Based on the results, the number of intestinal organoids per basement matrix dome was highest in location #1 ( Figure 2B), indicating the most growth potential for the derivation of intestinal organoids. In addition, to identify intestinal stem cells in vivo, immunohistochemistry with respect to several markers involved in intestinal stem cells and epithelial cells was conducted. ...
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... addition, to identify intestinal stem cells in vivo, immunohistochemistry with respect to several markers involved in intestinal stem cells and epithelial cells was conducted. As shown in Figure 2C, intestinal crypts isolated from location #1 of the small intestine had a distinct expression, such as leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a key gene required for stemness that is expressed in columnar crypt cells, B lymphoma Mo-MLV insertion region 1 homology (Bmi1), which was found in +4 cells adjacent to Paneth cells and F-actin in the intestinal epithelial cytoskeleton. Moreover, the fluorescently stained crypts showed epithelium-specific expression of Mucin2 in goblet cells, Ecadherin in adherent junctions (Supplementary Figure S3). ...
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... intestinal tissue sections were subjected to anatomical analysis using haematoxylin and eosin histological staining to identify distinct crypt and villus structures. As shown in Figure 2A, locations #1 and #2 were better developed than locations #3 and #4. The detailed view from vertical and horizontal sections in locations #1 and #2 showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side (Supplementary Figure S2). ...
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... shown in Figure 2A, locations #1 and #2 were better developed than locations #3 and #4. The detailed view from vertical and horizontal sections in locations #1 and #2 showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side (Supplementary Figure S2). Furthermore, to verify the efficiency of the derivation of intestinal organoids from four different locations, we subsequently cultivated intestinal organoids. ...
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... to verify the efficiency of the derivation of intestinal organoids from four different locations, we subsequently cultivated intestinal organoids. Based on the results, the number of intestinal organoids per basement matrix dome was highest in location #1 (Figure 2B), indicating the most growth potential for the derivation of intestinal organoids. In addition, to identify intestinal stem cells in vivo, immunohistochemistry with respect to several markers involved in intestinal stem cells and epithelial cells was conducted. ...
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... addition, to identify intestinal stem cells in vivo, immunohistochemistry with respect to several markers involved in intestinal stem cells and epithelial cells was conducted. As shown in Figure 2C, intestinal crypts isolated from location #1 of the small intestine had a distinct expression, such as leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a key gene required for stemness that is expressed in columnar crypt cells, B lymphoma Mo-MLV insertion region 1 homology (Bmi1), which was found in +4 cells adjacent to Paneth cells and F-actin in the intestinal epithelial cytoskeleton. Moreover, the fluorescently stained crypts showed epithelium-specific expression of Mucin2 in goblet cells, E-cadherin in adherent junctions (Supplementary Figure S3). ...
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... addition, we investigated the paracellular permeability character of the epithelial layer using fluorescent tracers up to 4 hr after treatment. FITC-dextran labelled the organoid lumen, demonstrating a high permeability for compounds of up to 4 kDa, such as glucose, peptides, and Figure 2. Immunohistochemical analysis of bovine small intestine: (A) haematoxylin and eosin histological staining to identify distinct crypt and villus structures from four different locations (#1, #2, #3, and #4) in the jejunum between the duodenum and ileum. ...
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... to assess the position effects for efficient isolation and derivation of intestinal organoids along the length of the small intestine in bovine, we sectioned four different locations in the jejunum between the duodenum and ileum. Interestingly, we found that jejunum (location #1 and #2) close to the duodenum showed integral structures of the intestinal epithelium gland, such as crypts at the bottom and finger-shaped villi on the apical side and intestinal crypts isolated from location #1 in the jejunum close to the duodenum had the most growth potential for the derivation of intestinal organoids (Figure 2). However, mouse models have shown that more distal tissues in the foetal intestine can be formed organoids well [37], suggesting the regional differences in vitro growth potential. ...
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... organoids show various developmental stages and can be spheroidal (round shaped), show budding (spheroids with extension), and have mature villus and crypt-like structures (branched structures). The detailed structures represent organoid propagation from day 2 to the fully grown structure on day 8. Scale bar: 100 µm (day 0, day 2) and 50 µm (day 3, day 6, and day 8), Figure S2: Histological H and E staining of the vertical and horizontal views reveal the positions of the intestinal epithelial glands, such as crypts (oval structures at the bottom) and villi (finger-like structures) cells, with varying magnification (40×, 100×, and 200×) at four different locations from #1 to #4. The purple spots show nuclei and the epithelium is stained pink, Figure S3: immunohistochemistry of Bmi1, E-cadherin, Chromogranin A, and Muccin2 in bovine small intestine. ...
Citations
... Relative expression levels of target genes were determined by using GAPDH, RPL0, and ACTB as the internal control [41][42][43] and compared between control and EHEC-infected monolayers. Primers used in this study were adopted from previous studies and summarized in Table 1 [42][43][44][45]. RT-qPCR reactions were carried out in duplicate from three biological replicates with two technical replicates per experiment. ...
Enterohemorrhagic Escherichia coli (EHEC) is a critical public health concern due to its role in severe gastrointestinal illnesses in humans, including hemorrhagic colitis and the life-threatening hemolytic uremic syndrome. While highly pathogenic to humans, cattle, the main reservoir for EHEC, often remain asymptomatic carriers, complicating efforts to control its spread. Our study introduces a novel method to investigate EHEC using organoid-derived monolayers from adult bovine ileum and rectum. These polarized epithelial monolayers were exposed to EHEC for four hours, allowing us to perform comparative analyses between the ileal and rectal tissues. Our findings mirrored in vivo observations, showing a higher colonization rate in the rectum compared with the ileum (44.0% vs. 16.5%, p < 0.05). Both tissues exhibited an inflammatory response with increased expression levels of TNF-a (p < 0.05) and a more pronounced increase of IL-8 in the rectum (p < 0.01). Additionally, the impact of EHEC on the mucus barrier varied across these gastrointestinal regions. Innovative visualization techniques helped us study the ultrastructure of mucus, revealing a net-like mucin glycoprotein organization. While further cellular differentiation could enhance model accuracy, our research significantly deepens understanding of EHEC pathogenesis in cattle and informs strategies for the preventative measures and therapeutic interventions.
... This consistently obtained from healthy donors, improved cell viability, and greater cellular diversity achievable only with non-immortalized tissue. Comparative tissue transcriptomics and characterization of intestinal organoids reveal similarities in conserved orthologous genes and cellular potentials between humans and cattle 18 . Therefore, a bovine organoidderived culture system may be advantageous in investigating human intestinal diseases, with findings easily translatable to human medicine. ...
... The small intestine plays a crucial role in animals in maintaining homeostasis via interacting with the microbiome and improving productivity in many physiological events, such as nutrient absorption, hormone secretion, and host-pathogen interactions from various intestinal cell types, including Paneth cells, enteroendocrine cells, goblet cells, and enterocytes (Olayanju et al., 2019;. As a potential alternative to an in vivo system, intestinal organoids, have attracted much attention for studying various functions of the intestinal epithelium and creating new possibilities and opportunities for testing as an in vitro model instead of animal testing (Rallabandi et al., 2020;Lee et al., 2021;. ...
... Organoids mimic the functionality and organization of organ tissues and have become an invaluable tool for the in vitro study of biological processes, development, and diseases [13][14][15][16]. In the field of livestock biotechnology, organoid-based systems have been used for disease modeling, the determination of host pathogens interactions, and nutritional research to improve the productivity of livestock [17][18][19]. Organoids can be created from adult stem cells, pluripotent stem cells, embryonic stem cells, and intestinal stem cells (ISCs) when cultured under the appropriate conditions. These cells undergo self-renewal, morphogenesis, self-organization, and differentiation within the crypt villus domain [20,21]. ...
... These results may indicate that 4 kDa FITC-dextran can enter into the lumen because of time effect. On the other hand, a few previous results showed that FITC dextran can enter into the lumen within 30 min [17,38,39]. These results may be attributed to differences in the culture condition including composition of the medium. ...
Background
Deoxynivalenol (DON) is a mycotoxin that has received recognition worldwide because of its ability to cause growth delay, nutrient malabsorption, weight loss, emesis, and a reduction of feed intake in livestock. Since DON-contaminated feedstuff is absorbed in the gastrointestinal tract, we used chicken organoids to assess the DON-induced dysfunction of the small intestine.
Results
We established a culture system using chicken organoids and characterized the organoids at passages 1 and 10. We confirmed the mRNA expression levels of various cell markers in the organoids, such as KI67 , leucine-rich repeat containing G protein-coupled receptor 5 ( Lgr5 ), mucin 2 ( MUC2 ), chromogranin A ( CHGA ), cytokeratin 19 ( CK19 ), lysozyme ( LYZ ), and microtubule-associated doublecortin-like kinase 1 ( DCLK1 ), and compared the results to those of the small intestine. Our results showed that the organoids displayed functional similarities in permeability compared to the small intestine. DON damaged the tight junctions of the organoids, which resulted in increased permeability.
Conclusions
Our organoid culture displayed topological, genetic, and functional similarities with the small intestine cells. Based on these similarities, we confirmed that DON causes small intestine dysfunction. Chicken organoids offer a practical model for the research of harmful substances.
... A bovine intestinal epithelial cell line has been established in Japan and used to test probiotic strains [65]. Moreover, 2D and 3D bovine intestinal organoid systems have recently been developed [66,67]. Thus, it would be of great interest for future studies to evaluate the performance of L. animalis 506 or other probiotic strains or strain combinations in similar assays using either of these model systems. ...
This study investigated the impact of L. animalis 506 on gut barrier integrity and regulation of inflammation in vitro using intestinal epithelial cell lines. Caco-2 or HT29 cell monolayers were challenged with enterotoxigenic E. coli (ETEC) or a ruminant isolate of Salmonella Heidelberg in the presence or absence of one of six probiotic Lactobacillus spp. strains. Among these, L. animalis 506 excelled at exerting protective effects by significantly mitigating the decreased transepithelial electrical resistance (TEER) as assessed using area under the curve (AUC) (p < 0.0001) and increased apical-to-basolateral fluorescein isothiocyanate (FITC) dextran translocation (p < 0.0001) across Caco-2 cell monolayers caused by S. Heidelberg or ETEC, respectively. Similarly, L. animalis 506 and other probiotic strains significantly attenuated the S. Heidelberg- and ETEC-induced increase in IL-8 from HT29 cells (p < 0.0001). Moreover, L. animalis 506 significantly counteracted the TEER decrease (p < 0.0001) and FITC dextran translocation (p < 0.0001) upon challenge with Clostridium perfringens. Finally, L. animalis 506 significantly attenuated DON-induced TEER decrease (p < 0.01) and FITC dextran translocation (p < 0.05) and mitigated occludin and zona occludens (ZO)-1 redistribution in Caco-2 cells caused by the mycotoxin. Collectively, these results demonstrate the ability of L. animalis 506 to confer protective effects on the intestinal epithelium in vitro upon challenge with enteric pathogens and DON known to be of particular concern in farm animals.
... Understanding host-pathogen interactions for these zoonotic enteric pathogens requires consideration of the specifc gut segments they inhabit [13,14]. Although intestinal organoid technology has been described for certain segments of the bovine gastrointestinal tract (i.e., the jejunum [5,15], ileum [16,17] and colon [18]), there remains a gap in our understanding of the duodenum and rectum. Addressing this gap is crucial for advancing our knowledge of pathophysiology and colonization processes. ...
... To bridge these knowledge gaps and enhance our understanding of bovine gut physiology and pathogen interactions, we present a technique for culturing bovine intestinal organoids from fve diferent segments of the adult bovine gut. Tis method, distinct from previous fullthickness tissue sampling approaches [4,5,7,[15][16][17][18], involves mucosa extraction using biopsy forceps and subsequent organoid cultivation with an in-house organoid growth media supplemented with growth factors and inhibitors. Te resulting organoids are assessed for their fdelity to in vivo bovine gut traits through immunofuorescence and RT-qPCR analysis. ...
... intestinal organoids were successfully generated from fresh tissue samples obtained via the biopsy technique from the duodenum, jejunum, ileum, colon, and rectum of fve adult cattle (Figure 2(a)). Organoids generally formed spherical or budding luminal structures consistent with previous reports [5,[15][16][17][18]28]. Te success rate of initial organoid development using this biopsy technique was 100% in all the fve segments ( Table 2). ...
Recent progress in bovine intestinal organoid research has expanded opportunities for creating improved in vitro models to study intestinal physiology and pathology. However, the establishment of a culture condition capable of generating organoids from all segments of the cattle intestine has remained elusive. Although previous research has described the development of bovine jejunal, ileal, and colonic organoids, this study marks the first report of successful bovine duodenal and rectal organoid development. Maintenance of these organoids through serial passages and cryopreservation was achieved, with higher success rates observed in large intestinal organoids compared to their small intestinal counterparts. A novel approach involving the use of biopsy forceps during initial tissue sampling streamlined the subsequent tissue processing, simplifying the procedure compared to previously established protocols in cattle. In addition, our study introduced a more cost-effective culture medium based on advanced DMEM/F12, diverging from frequently used commercially available organoid culture media. This enhancement improves the accessibility to organoid technology by reducing culture costs. Crucially, the derived organoids from the jejunum, ileum, colon, and rectum faithfully preserved the structural, cellular, and genetic characteristics of the in vivo intestinal tissue. This research underscores the significant potential of adult bovine intestinal organoids as a physiologically and morphologically relevant in vitro model. Such organoids provide a renewable and sustainable resource for a broad spectrum of studies, encompassing investigations into normal intestinal physiology in cattle and the intricate host-pathogen interactions of clinically and economically significant enteric pathogens.
... ET release and bacterial killing were evaluated along with cytokine secretion offering information on the mechanisms that operate in the defense of these cell types against MAP. Protocols for culturing ruminant organoids from different digestive segments have been set out [112][113][114][115][116][117][118][119][120]. Since the first report in 2009, intestinal organoids have evolved as a potential alternative to in vivo models for various experimental purposes, such as imaging, molecular analysis and gene editing, but also as reductionist approaches to study the interaction of epithelial cells with other relevant actors in the physiological context, such as immune cells [121,122] or microbiota [123,124] or even to evaluate the early host-pathogen interaction in the context of many infectious diseases (protozoa: [125,126]; virus: reviewed in [127]; bacterias: [128][129][130][131][132][133]). ...
Paratuberculosis (PTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects a wide variety of domestic and wild animals. It is considered as one of the diseases with the highest economic impact in the ruminant industry. Despite many efforts and intensive research, PTB control is still controversially discussed and diagnostic and immunoprophylactic tools lack great limitations. Thus, models play a crucial role in understanding the pathogenesis of infection and disease, and in testing novel vaccine candidates. Here, we review the potential and limitations of different experimental approaches currently used in PTB research, focusing on laboratory animals and cell based models. The aim of this review is to offer a vision of the models that have been used and what has been achieved or discovered with each one so that the reader can choose the best model to answer their scientific questions and prove their hypotheses. Also, we bring forward new approaches that we consider worth exploring in the near future.
... The gene expression of intestinal stem cells (LGR5+) and differentiation markers (ChrA and Muc2) were analysed using qPCR followed by immunostaining to look for proteins (ChrA, Muc2, sucrase isomaltase, and lysozyme). A panel of three commonly used housekeeping genes (18s rRNA, GAPDH, and ACTB) were tested [31][32][33], to normalize the gene expression. ...
... The immune genes included in the present study were selected as they had been shown to be differentially expressed with a high fold change at 72 hpi during BCoV infection of HCT-8 cells [30]. A panel of three commonly used housekeeping genes (18s rRNA, GAPDH, and ACTB) were tested [31][32][33], and the most stably expressed (similar Ct-values in mockand BCoV-infected enteroids) were included to normalize the gene expression results. All primers are listed in Table S3 [40][41][42][43]. ...
Bovine coronavirus (BCoV) is one of the major viral pathogens of cattle, responsible for economic losses and causing a substantial impact on animal welfare. Several in vitro 2D models have been used to investigate BCoV infection and its pathogenesis. However, 3D enteroids are likely to be a better model with which to investigate host–pathogen interactions. This study established bovine enteroids as an in vitro replication system for BCoV, and we compared the expression of selected genes during the BCoV infection of the enteroids with the expression previously described in HCT-8 cells. The enteroids were successfully established from bovine ileum and permissive to BCoV, as shown by a seven-fold increase in viral RNA after 72 h. Immunostaining of differentiation markers showed a mixed population of differentiated cells. Gene expression ratios at 72 h showed that pro-inflammatory responses such as IL-8 and IL-1A remained unchanged in response to BCoV infection. Expression of other immune genes, including CXCL-3, MMP13, and TNF-α, was significantly downregulated. This study shows that the bovine enteroids had a differentiated cell population and were permissive to BCoV. Further studies are necessary for a comparative analysis to determine whether enteroids are suitable in vitro models to study host responses during BCoV infection.
... The small intestine consists of the duodenum, jejunum, and ileum and performs a variety of functions, such as nutrient absorption, electrolyte uptake, hormone secretion and host-pathogen interactions in intestinal epithelium that is composed of a variety of intestinal cell types such as Paneth cells, enteroendocrine cells, goblet cells and enterocytes [1,2]. Recently, a scaffold-based three-dimensional (3D) culture system has provided a reliable alternative platform for the establishment of pluripotent and adult stem cells derived intestinal organoids in vitro. ...
... Recent progress has been made to establish intestinal organoids derived from livestock, including bovine [2], porcine [4], chicken [5] and equine [6]. In particular, we previously reported a reliable method for the isolation of intestinal crypts from small intestines and the robust 3D expansion of intestinal organoids (basal-out) derived from adult bovine [2,7]. ...
... Recent progress has been made to establish intestinal organoids derived from livestock, including bovine [2], porcine [4], chicken [5] and equine [6]. In particular, we previously reported a reliable method for the isolation of intestinal crypts from small intestines and the robust 3D expansion of intestinal organoids (basal-out) derived from adult bovine [2,7]. Genearally, basal-out intestinal oragnoids have the intestianl basal membrane on the surface. ...
Recently, we reported the robust in vitro three-dimensional (3D) expansion of intestinal organoids derived from adult bovine (> 24 months) samples. The present study aimed to establish an in vitro 3D system for the cultivation of intestinal organoids derived from growing cattle (12 months old) for practical use as a potential alternative to in vivo systems for various purposes. However, very few studies on the functional characterization and 3D expansion of adult stem cells from livestock species compared to those from other species are available. In this study, intestinal crypts, including intestinal stem cells, from the small intestines (ileum and jejunum) of growing cattle were isolated and long-term 3D cultures were successfully established using a scaffold-based method. Furthermore, we generated an apical-out intestinal organoid derived from growing cattle. Interestingly, intestinal organoids derived from the ileum, but not the jejunum, could be expanded without losing the ability to recapitulate crypts, and these organoids specifically expressed several specific markers of intestinal stem cells and the intestinal epithelium. Furthermore, these organoids exhibited key functionality with regard to high permeability for compounds up to 4 kDa in size (e.g., fluorescein isothiocyanate [FITC]-dextran), indicating that apical-out intestinal organoids are better than other models. Collectively, these results indicate the establishment of growing cattle-derived intestinal organoids and subsequent generation of apical-out intestinal organoids. These organoids may be valuable tools and potential alternatives to in vivo systems for examining host-pathogen interactions involving epithelial cells, such as enteric virus infection and nutrient absorption, and may be used for various purposes.
... Further, organoids can be generated from tissue samples from both healthy and diseased animals. Thus, they can effectively model both normal and pathological conditions in in vitro environment (10)(11)(12). At present, development of farm and companion animal organoids has only been reported using ASCs (2). ...
Animal organoid models derived from farm and companion animals have great potential to contribute to human health as a One Health initiative, which recognize a close inter-relationship among humans, animals and their shared environment and adopt multi-and trans-disciplinary approaches to optimize health outcomes. With recent advances in organoid technology, studies on farm and companion animal organoids have gained more attention in various fields including veterinary medicine, translational medicine and biomedical research. Not only is this because three-dimensional organoids possess unique characteristics from traditional two-dimensional cell cultures including their self-organizing and self-renewing properties and high structural and functional similarities to the originating tissue, but also because relative to conventional genetically modified or artificially induced murine models, companion animal organoids can provide an excellent model for spontaneously occurring diseases which resemble human diseases. These features of companion animal organoids offer a paradigm-shifting approach in biomedical research and improve translatability of in vitro studies to subsequent in vivo studies with spontaneously diseased animals while reducing the use of conventional animal models prior to human clinical trials. Farm animal organoids also could play an important role in investigations of the pathophysiology of zoonotic and reproductive diseases by contributing to public health and improving agricultural production. Here, we discuss a brief history of organoids and the most recent updates on farm and companion animal organoids, followed by discussion on their potential in public health, food security, and comparative medicine as One Health initiatives. We highlight recent evolution in the culturing of organoids and their integration with organ-on-a-chip systems to overcome current limitations in in vitro studies. We envision multidisciplinary work integrating organoid culture and organ-on-a-chip technology can contribute to improving both human and animal health.
