Assessment of Intestinal Permeability in Young Piglet to Pave the Way to Oral Vaccination

The small intestine of the piglet has evolved to be permeable immediately after birth to facilitate the uptake of colostrum-derived immunoglobulins, other macromolecules, and cells. However, the precise timing of gut closure in today’s precocious pig is not known. We gavaged piglets immediately after birth and at 1 hour after birth with Cy5-labeled Ovalbumin (Cy5-Ova) then harvested their small intestine’s 6-7 hours later. To assess localization of Cy5-Ova in the small intestinal epithelial cells, we perform immunohistochemistry using a basolateral surface marker and a recycling endosome marker called pIgR, the late endosomal marker Rab7, and the lysosomal marker LAMP-1. Cy5-Ova co-localized with Rab7 and LAMP-1 in the duodenum and jejunum of 0 hour old and 1 hour old gavaged piglets, but only in the ileum of 0 hour gavaged piglets. These data suggest that movement of Cy5-Ova through the late endosomes to the lysosomes was much reduced in the ileum of 1 hour gavaged piglets, possibly impacted by prior processing of colostral macromolecules. Cy5-Ova was largely present in epithelial cell digestive and transport vacuoles, but it did not colocalize with pIgR-positive endosomes in 0 hour and 1 hour gavaged piglets. Understanding the relationship between the localization of Cy5-Ova and small intestinal permeability may contribute to establishing whether oral vaccination in the newborn can capitalize on the transient permeability before gut closure to promote immune protection.


Introduction
Fetal and newborn piglet intestinal enterocytes possess an apical canalicular system which allows for the production of cytoplasmic vacuoles of various sizes, which are vital for colostrum uptake (Skrzypek et al., 2018). These fetal-derived enterocytes have large vacuoles (leading to these cells being named vacuolated fetal enterocytes (VFEs)) that absorb and transport macromolecules either to the basolateral surface where they are expelled, or to the lysosomes where they are digested (Skrzypek et al., 2018;Skrzypek et al., 2007). VFEs are rst formed in the duodenum in the pig fetus. In the second trimester of pregnancy, VFEs become redistributed towards the jejunal and ileal regions of the small intestine (Olszewski et al., 2021;Smith and Jarvis, 1978). VFEs can non-selectively absorb high molecular weight substances by pinocytosis or endocytosis at the apical area of the enterocyte (Fujita et al., 2007;Michael Danielsen and Hansen, 2016) but only for a short time after birth (Salmon, 2012;Sangild, 2003). VFEs are comprised of giant transport vacuoles that disappear 2-3 days after birth and giant digestive vacuoles that are present for up to 3 weeks of age (Baintner, 1994;Baintner, 2007) until VFEs are replaced by adulttype enterocytes that lack an apical canicular system (Skrzypek et al., 2007). Transport vacuoles are formed immediately apical to the nucleus of the enterocyte after newborn piglets consume colostrum and the macromolecules are taken up via endocytosis. The transport vacuoles then migrate to the basolateral area of the cell where the majority of macromolecules, including IgG, completely bypass the Golgi cisternae to release the luminal substances into the intercellular space via exocytosis with preservation of their biological activity (Baintner, 1994;Baintner, 2007;Burton and Smith, 1977;Rodewald and Kraehenbuhl, 1984;Zabielski, 1998). In contrast, digestive vacuoles are relatively large, formed near the apical regions of the cell, and do not migrate (Baintner, 1994;Baintner, 2007). These digestive vacuoles contain nutrients from colostrum and milk that are decomposed into their base components due to enzymes released by connecting lysosomes (Baintner, 1994).
Enterocytes have a basolateral and an apical domain which is critical for epithelial cell homeostasis and function. Cellular homeostasis is dependent on the internalization of small solutes, macromolecules, and plasma membrane receptors driven by endocytosis. Endocytosis is mediated by a complex interplay of Rab GTPases that function by regulating epithelial membrane tra cking as well as tethering and budding of vesicles at different locations within epithelial cells (Gillingham et al., 2014;Homma et al., 2019). Rab7 regulates late endosomal membrane fusion and tra cking in the perinuclear region via the interaction of Rab7-RILP-dyenin-dynactin for the biogenesis and maintenance of the lysosomal compartment (Zhang et al., 2009).
Lysosomes are the terminal degradative compartments of cells and they contain hydrolytic enzymes such as acid hydrolases that degrade cell debris into precursor molecules for macromolecule synthesis.
Lysosomal-associated membrane protein 1 (LAMP-1) is a highly N-glycosylated protein which is transported from the trans-Golgi network to lysosomes via endosomes (Wilke et al., 2012;Xu et al., 2012).
Polymeric immunoglobulins (pIgs) are synthesized from lamina propria plasma cells in the small intestine. They contain a J-chain and a small acidic polypeptide which connects two IgAs to form dimeric IgA, also known as pIgA (Asano and Komiyama, 2011;Strugnell and Wijburg, 2010). pIgA binds to the transmembrane pIgR on the basolateral surface of the polarized intestinal epithelial cell (IEC) and the pIgR-pIgA complex is internalized into the basolateral early endosome followed by the microtubuledependent delivery of the pIgR-pIgA complex to the common recycling endosome (CRE) (Verges, 2016;Verges et al., 2004). The pIgR-pIgA complex then travels to the apical surface of the cell within a series of tubules and vesicles from specialized subdomains of the CRE where it fuses with the apical plasma membrane and is expelled (Strugnell and Wijburg, 2010;Verges, 2016;Verges et al., 2004).
We intended to discern the localization of Cyanine-5 labeled ovalbumin (Cy5-Ova) consumed immediately after birth (0 hour old gavaged piglets) and 1 hour after birth (1 hour old gavaged piglets) using a series of markers pertaining to polarized IECs including pIgR (basolateral surface or endosome marker), Rab7 (late endosomal marker), and LAMP-1 (lysosomal marker). The 0 hour and 1 hour time points were chosen to assess whether the presence of ingested colostrum affected antigen uptake and/or localization within the small intestine.

Animal Use and Ethics
This work was approved by the University of Saskatchewan's Animal Research Ethics Board and adhered to the Canadian Council on Animal Care Guidelines for humane animal use.

Labeling of Ova with Cy5
Ovalbumin from chicken egg white (Ova) (Sigma-Aldrich Canada Ltd, Oakville, ON; A5503) was labeled with Cyanine-5 (Cy5) reactive dye (Ambion/ThermoFisher Scienti c, Burlington, ON, Canada; 5831G). The following formula was used to determine the amount of Cy5 needed for labeling: 8 x molecular weight (MW) of Cy5 x (amount of Ova)/ MW of Ova. Each Cy5 tube was re-suspended in 100 ml of dimethyl sulfoxide (DMSO; Sigma-Aldrich; D2650). A 1:10 ratio of Cy5 dye to protein and 0.3 M sodium carbonate buffer (Sigma-Aldrich) were incubated overnight at 4°C with nutation and then placed on top of a 3K Amicon centrifugal lter (ThermoFisher Scienti c) before centrifugation at 16,000 x g for 10 minutes.
After centrifugation, lters were inverted, and samples were washed 4 times with distilled water. The lters were then placed in new microcentrifuge tubes and then centrifuged at 1000 x g for 2 minutes to dispense the Cy5-labeled Ova.

Degree of Labeling of Cy5 to Ova
To calculate the degree of labeling of Cy5 to Ova, absorbance values of Cy5 at A 280 and A 555 were rst calculated with a Biochrom Spectrophotometer (Libra S22, MBI Lab Equipment, Kirkland, QC). Next, the protein concentration was calculated using the molar extinction coe cient of Ova. Finally, the moles of Cy5 per moles of Ova were calculated using the molar extinction coe cient of Cy5.

Tissue Collection
Piglets were randomly selected from 3 litters immediately after birth, marked, and fed 300 mg of Cy5-Ova suspended in a total volume of 14.2 ml phosphate-buffered saline (PBS; Sigma-Aldrich) with a gavage tube gently inserted into their stomachs (termed "0 hour old gavaged", n=3, one per litter). Other piglets were marked immediately after birth; however, they were not fed 300 mg of Cy5-Ova until 1 hour after birth (termed "1 hour old gavaged", n=3, one per litter). A 1 hour old control piglet that was not gavaged with Cy5-Ova was also selected. All piglets were left to suckle from their sows for another 5 hours. The piglets were then were humanely euthanized by a non-penetrating Zephyr machine coupled with exsanguination.
Blood serum, stomach contents, contents of initial gavage (Cy5-Ova (300 mg/14.2 mL)), and distilled water (control) were collected and uorescent intensity of Cy5 (arbitrary units) was measured with an Odyssey infrared imager (LI-COR Biosciences, Lincoln, NE) at 700 nm. Duodenum, jejunum, and ileum intestinal segments (10-15 cm in length) were obtained and placed within separate 100 ml Erlenmeyer asks with DMEM media (Sigma-Aldrich). Serum, stomach contents, and urine samples were collected from all piglets. Small cross-sections (1 cm 2 ) of the intestinal segments from each piglet were placed within tissue-loc biopsy cassettes (ThermoFisher Scienti c; 58931) for immunohistochemistry (IHC) purposes. Tissues were xed in 10% buffered formalin (Sigma-Aldrich) for 48 hours and then dehydrated for 24 hours in a series of increasing concentrations of alcohol (EtOH 70%, EtOH 80%, EtOH 95%, EtOH 100%, EtOH 100% -xylene equal mix, and xylene) with the use of a RVG1 tissue processor (Rankin, MI, USA) before embedding in para n. Once tissues were embedded in para n blocks, a Microm Automatic Microtome (Thermo Scienti c) was used to cut 5 mm tissue slices which were carefully placed on Superfrost Plus microscope slides (ThermoFisher Scienti c; 22-034-979) before dehydration at 60 °C overnight.

Immunohistochemistry
Small intestinal tissue sections of 0 hour old gavaged and 1 hour old gavaged piglets were removed from the oven and de-para nized in decreasing concentrations of alcohol (xylene, EtOH 100%, EtOH 95%, and EtOH 70%). Slides were blocked for 3 hours at room temperature in 5% (w/v) blotting grade blocker nonfat dry milk (

Fluorescence Quanti cation
The uorescence of all IHC images was quanti ed using Fiji software (a distribution of ImageJ software).
The corrected total cell uorescence (CTCF) was measured in speci c regions of interest (ROIs) in each image. CTCF = integrated density -(area of the selected region X mean uorescence of background).

Statistics
Differences between the 0 hour old and 1 hour old gavaged animals as well as the control animals were analyzed using Kruskal-Wallis One-way analysis of variance (ANOVA) test with Dunn's multiple comparison's test. Differences were considered statistically signi cant if P < 0.05.

Fluorescent Intensity of Cy5 in Different Bodily Compartments
The uorescent intensity (arbitrary units) of Cy5 was calculated in different bodily uids of 0 hour old gavaged and 1 hour old gavaged piglets ( gure 1). The uorescent intensity of Cy5 in serum and the stomach of 0 hour old and 1 hour old gavaged piglets appear to be relatively consistent and low relative to the input ( gure 1). No Cy5 uorescence was detected in distilled water (control) for both groups, as expected ( gure 1). The presence of Cy5-Ova within serum of 0 hour old gavaged and 1 hour old gavaged piglets suggests that the small intestinal epithelium is permeable to proteins immediately after birth.

Zero Hour Old Gavaged and 1 Hour Old Gavaged Piglets
Para n-embedded immunohistochemistry (IHC-p) was performed on intestinal tissue from 0 hour old gavaged piglets and 1 hour old gavaged piglets to visualize region-speci c differences of duodenal, jejunal, and ileal IECs basolateral surface or endosome marker (anti-pIgR), late endosomal marker (anti-Rab7), and lysosomal marker (anti-LAMP-1), and to localize Cy5-Ova within the IECs. To assess any uorescence due to non-speci c binding of the secondary antibodies and to assess background uorescence in the uorescence range for Cy5 dye, intestinal tissues from a 1 hour old control piglet that was not gavaged with Cy5-Ova were incubated with Alexa 555-labeled goat anti-rabbit IgG (green; secondary antibody for anti-pIgR, gure 2a-c), anti-mouse IgG2b labeled-FITC (blue; secondary antibody for anti-Rab7, gure 2d-f), and anti-mouse IgG1 labeled-FITC (blue; secondary antibody for anti-LAMP-1, gure 2g-i). In gures 2a-c, there is a very weak, green uorescent signal in the lamina propria regions indicating very weak background uorescence or non-speci c binding of the Alexa 555-labeled goat-antirabbit IgG secondary antibody. In gures 2d-f, there is a very weak blue uorescent signal indicating very weak background uorescence or non-speci c binding of the anti-mouse IgG2b labeled-FITC secondary antibody, again within the lamina propria. In gures 2g-I, there is a very weak blue uorescent signal at the basolateral surface and the lamina propria region indicating very weak background uorescence or non-speci c binding of the anti-mouse IgG1 labeled-FITC secondary antibody.
In gures 2j-l, we quanti ed the uorescence at 633 nm which corresponds to Cy5 uorescence, to discern background uorescence. There appeared to be weak uorescence in the lamina propria, especially in the duodenal region (Fig 2j) with uorescence in the endosomes in the jejunum (Fig 2k) and ileum (2L).
We performed IHC-p to visualize the presence of pIgR, Rab7, LAMP-1, and Cy5-Ova within the duodenum of a 0 hour old gavaged piglets. In gure 3a, pIgR apepars located within small, medium, and large-sized transport and digestive vacuoles with strong uorescence throughout the duodenal intestinal epithelium (white circle). Cy5-Ova is located throughout the cell in tiny endsomes most noticably between duodenal IECs (purple arrow) and basolaterally (orange arrow) with moderate uorescence ( gure 3b) with no merging of uorescnece with pIgR ( g 3c). Rab7 is present within very small endosomes throughout duodenal IECs (white circle) near the lateral surface between cells (purple arrow) as well as on the apical surface of cells independent of endosomes (white arrow; gure 3d). Cy5-Ova is located within large vacuoles in this duodenal section as well as in endosomes throughout the cells ( gure 3e) where it colocalizes with Rab7 ( gure 3f). In gure 3e, Cy5-Ova also appears to be located within lamina propria cells (orange circle) with moderate to strong uorescence. LAMP-1 is located within lysosomes throughout duodenal IECs (white circle) as well as in lysosomes near the lateral surface between cells (purple arrow). LAMP-1 also appears to be located in lysosomes just beneath the apical surface of the cells (white arrow) ( gure 3g,i). In gure 3e, Cy5-Ova is located within small, medium, and large-sized transport and digestive vacuoles and in some lysosomes with moderate uorescence (white circle) where it colocalizes with LAMP-1 (3h).
We next assessed the presence of pIgR, Rab7, LAMP-1, and Cy5-Ova within the jejunum of a 0 hour old gavaged piglet. In gure 4a, pIgR is located throughout the jejunal IEC endosomes (large white circle), on the apical surface (white arrow) as well as within medium-sized transport and digestive vacuoles near the basolateral surface (small white circle) of the cells. In gure 4b, Cy5-Ova is located on the apical surface (white arrow) of cells as well as within medium-sized transport and digestive vacuoles near the basolateral surface of the cells (white circle) independent of pIgR ( gure 4c). In gure 4d, Rab7 is located in endosomes just below the apical surface (white arrow) of jejunal IECs with minimal expression as well as within endosomes throughout the cells (white circle). Cy5-Ova is colocalized with Rab7 within endosomes throughout the jejunal IECs (white circle; gure 4e-f). LAMP-1 ( gure 4g) and Cy5-Ova ( gure 4h) are colocalized within lysosomes throughout jejunal IECs (white circles), however, some lysosomes also appear to be independent of Cy5-Ova ( gure 4f).
Next, we probed for the presence of pIgR, Rab7, LAMP-1 in the small intestine 6 hours post gavage when piglets were gavaged with Cy5-Ova 1 hour after birth. In gure 6a, pIgR is located within small to mediumsized transport and digestive vacuoles with strong uorescence throughout the duodenal intestinal epithelium (large white circle) as well as within small endosome (small white circle) and within lamina propria cells (orange circles). In gure 6b, Cy5-Ova is located within lamina propria cells (orange circle) of the duodenal intestinal epithelium with weak to moderate uorescence. In gure 6d, Rab7 is located on the basolateral surface (orange arrow) throughout the cells (white circle) of duodenal IECs. In gure 6e, Cy5-Ova is located within large-sized digestive vacuoles with moderate to strong uorescence and with some evidence of colocalization with Rab7 in endosomes throughout the cell (Fig 6f). In gure 6g, LAMP-1 is located within lysosomes throughout the duodenal IECs (white circle). In gure 6h, Cy5-Ova appears to be located within small and medium-sized transport and digestive vacuoles with moderate to strong uorescence as well colocalized within lysosomes (6i). Cy5-Ova is also present within duodenal lamina propria cells (orange circle ; Fig 6h).
When we investigated the jejunum of 1 hour old gavaged piglets, pIgR is located within small, medium, and large-sized transport and digestive vacuoles (Fig 7a, large white circle) throughout jejunal IECs with medium to strong uorescence. pIgR also appears to be located on the apical surface (white arrow) and throughout the cells within endosomes (small white circle) with weak uorscence. In gure 7b, Cy5-Ova is located primarily within jejunal lamina propria cells (orange circle), however, there appears to be several speckles of Cy5-Ova (small white circle) near the basolateral surface independent of pIgR (large white circle). In gure 7d, Rab7 is located on the apical surface independent of endsomes (white arrow) and located throughout jejunal IECs within endosomes (white circle). In gure 7e, Cy5-Ova appears to be located within small, medium, and large-sized transport and digestive vacuoles (white circle) throughout the cells largely independent of Rab7. In gure 7g, LAMP-1 appears located on the apical surface independent of lysosomes (white arrow) and located throughout ileal IECs within lysosomes (white circle). In gure 7h, Cy5-Ova appears to be located within small, medium, and large-sized transport and digestive vacuoles (white circle) throughout the cells largely independent of LAMP-1 (Fig 7i).
When we investigated the ileum of a 1 hour old gavaged piglet, we observed that pIgR is located within small to medium-sized vacuoles near the apical regions of ileal IECs (Fig 8a). In gure 8b, Cy5-Ova is located within large-sized transport and digestive vacuoles (white circle closest to the top of the image) that are irregular in shape in comparison to the typical circular nature of vacuoles. Cy5-Ova is also located within endosomes throughout the cells (white circle closest to the bottom of the image) independent of pIgR (Fig 8c). In gure 8d, Rab7 is located within endosomes near the lateral surface between ileal IECs (purple arrow). Rab7 ( gure 8d,f) and Cy5-Ova ( gure 8e,f) colocalize within endosomes throughout the cells (small white circle) and within medium to large-sized transport and digestive vacuoles (large white circles) that are an irregular shaped and located near the basolateral region of the cells. Finally, LAMP-1 appears to be located within lysosomes throughout ileal IECs (white circle ; Fig 8g). Cy5-Ova appears to be located within medium to large-sized transport and digestive vacuoles near the apical and basolateral regions of the cells (the two white circles closest to the right of the image; g 8h). Cy5-Ova is also located within lysosomes throughout the cells (white circle closest to the left of the image) with LAMP-1 (Fig 8i).
When we compare the corrected total cell uorescence with the 0 hour gavaged piglets, the 1 hour gavaged piglets and control animal piglet, we observed that the duodenum and jejunum from the 1 hour old gavaged piglets had signi cantly more pIgR, Rab7, and LAMP-1 (jejunum only) and Cy5-OVA (duodenum only) than the control animal (Fig 9a,b). In contrast, the ileum of the 0 hour old gavaged piglets had signi cantly more pIgR, Rab7 and Cy-5 relative the control tissue (Fig 9c).

Discussion
Piglets must ingest colostrum within hours after birth to receive colostrum-derived antibodies and macromolecules for immune system development and protection. It has been previously reported that the small intestine of newborn piglets that are cross-fostered take up colostrum-derived antibodies, macromolecules, and cells (Bandrick et al., 2014a;Bandrick et al., 2011;Bandrick et al., 2014b). However, colostral cells from a non-biological sow does not cross the suckling piglet's intestinal wall (Bandrick et al., 2011;Loving et al., 2014). Furthermore, all segments of the small intestine are reported to lose their abilities to transport macromolecules and cells across the intestinal epithelium 36 hours after birth (Sangild, 2003;Westrom et al., 1989). This loss of transport is also referred to as "gut closure" and is presumed to occur by decreased endocytotic capabilities of IECs, however, the exact mechanism is currently unknown. In addition, modi cation of the assembly and composition of tight junction proteins such as claudins which regulate the high-capacity pore pathway between IECs may also contribute to gut closure (Deluco et al., 2021;Pasternak et al., 2015a). Tight junctions are a complex of proteins that regulate the passage of small, uncharged solutes and ions between adjacent cells (Madara et al., 1992;Tsukita et al., 2001). Understanding the mechanism by which orally administered antigens traverse the intestinal epithelium immediately after birth may help to provide insight on the mechanism of uptake of an orally administered antigen that is relevant for vaccine development. Visualization of the location of the ingested antigen in relation to endosomal and epithelial cell surface markers may facilitate understanding of the mechanism of antigen transport and processing.
Throughout the small intestine, endocytosis takes place at the microvilli base where there are deep invaginations between the adjacent microvilli (Gonnella and Neutra, 1984). Research in the newborn rat ileum showed that enterocytes non-selectively take up macromolecules and process them through a complex array of membrane compartments towards a giant vacuole, which appears to be consistent with a lysosome that is responsible for degradation of milk-derived products (Knutton et al., 1974). Furthermore, macromolecules can be taken up by both receptor-mediated and non-selective endocytosis for degradation in the lysosome or for transport using the transepithelial transport pathway (Knutton et al., 1974;Siminoski et al., 1986). Others showed that radio-labeled proteins introduced to the rat jejunal and ileal epithelium localized to apical endosomal compartments and were also associated with lysosomal vacuoles, suggesting it was targeted for degradation and for transport but that they were also observed at basolateral cell surfaces and lamina propria suggesting transport (Gonnella et al., 1989;Gonnella et al., 1987). Understanding the mechanisms responsible for route the macromolecules towards transport in the neonatal period has implications for possibly manipulating barrier function.
In the present study, we investigated how Cy5-Ova is taken up and transported within IECs of the 0 hour old gavaged and 1 hour old gavaged piglet. In duodenal, jejunal, and ileal IECs, Cy5-Ova does not colocalize with pIgR on the surface of the cells or within pIgR+ endosomes, regardless of whether they were gavaged with Cy5-Ova pre-or post-suckling. One exception was that co-localization between pIgR and Cy5-Ova was observed within endosomes throughout ileal IECs in 0 hour old gavaged piglets. Cy5-Ova appeared to localize within small, medium, and large-sized transport and digestive vacuoles and largely colocalized with Rab7 in the endosomes throughout the small intestine in 0 hour gavaged piglets. In contrast, Cy5-Ova colocalized with Rab7 in the ileum but it was largely independent of Rab7 in the duodenum and jejunum in the 1 hour gavaged piglets. One limitation of this research may be the potential rupturing of vacuoles containing Cy5-Ova during IHC-p processing. During the dehydration process of aqueous formalin-xed tissues, both transport and digestive vacuoles located within IECs may have ruptured thus releasing their contents in the surrounding cytoplasmic areas. Our research shows the localization of Cy5-Ova within transport and digestive vacuoles located within small intestinal cells exhibit varying degrees of uorescent intensity. Areas in which vacuoles previously resided appear as black suggesting that these vacuoles may have contained Cy5-Ova but were 'washed out' despite the use of formaldehyde, a xing agent that is generally considered effective in cross-linking proteins and stabilizing the cell matrix.
Cy5-Ova colocalized with lysosomal marker LAMP-1 in duodenal, jejunal and ileal IECs in 0 hour gavaged piglets as well as being present witihin vacuoles. Colocalization with LAMP-1 was not observed within 1 hour old gavaged piglets. One possibility is that it takes longer for the piglets that received colostrum to process the gavaged Cy5-Ova and that, given more time, it would be present in the lysosomes in this region of the gut, or that there are region speci c differences in localization. Localization of Cy5-Ova within lysosomes of newborn piglets may indicate that the antigen is undergoing degradation and/or that we are simply observing cleavage of the Cy5 dye molecules from the antigen. SDS-PAGE analysis of the processed tissues may elucidate whether Ova is being cleaved from Cy5 in the lysosome or whether it remains covalently associated.
Colocalization observed between Rab7-Cy5-Ova and LAMP-1-Cy5-Ova suggests that Cy5-Ova has entered duodenal IECs via endocytosis and has progressed through early endsomes, recycling endosomes, late endosomes, and localized within lysosomes. To con rm the presence of Cy5-Ova within early endosomes and recycling endosomes, other markers should be explored such as Rab5 (early endosome marker) and Rab25 (recycling marker). Another future direction could be to examine the effects of endocytotic inhibitors on antigen uptake. Chloroquine is an aminoquinolone derivative that is used in the primary treatment of malaria. Chloroquine has also been shown to be an effective inhibitor of clathrin-dependent endocytosis by affecting the function of clathrin and clathrin-coated vesicles ). Filipin is a polyene antibiotic that binds to cholesterol within the epithelial cell membrane thus making it an effective inhibitor of clathrin-independent endocytosis (Dutta and Donaldson, 2012). Another technique to assess the localization of ingested antigen may include using BODIPY-conjugated DQ-Ova (a selfquenched conjugate of Ova) (Liu et al., 2017). Proteolytic cleavage of DQ-Ova in the lysosomes of newborn piglets would exhibit brighter uorescence due to the release of BODIPY dye molecules (Liu et al., 2017). The presence of our antigen within late endosomes and lysosomes suggests that Cy5-Ova is entering polarized IECs via endocytosis. Further analysis should be performed to determine whether an orally administered vaccine is degraded within the lysosomes and whether this negatively impacts antigen presentation and induction of the adaptive immune response.
In pigs, intestinal epithelial cells lack expression of MHCII molecules and therefore cannot act as APCs (Wilson et al., 1996). Therefore, it is critical that vaccines traverse the intestinal wall for oral vaccines to be presented and recognized by the adaptive immune system. Since the intestinal wall is semi-permeable in piglets at birth, this period of time may be used to orally vaccinate pigs, however, it is possible that the neonatal immune system may not be mature enough to respond to the oral vaccine. A previous study examining the effects of orally administered Ova with or without adjuvants in piglets within 6 hours of birth (Pasternak et al., 2015b) showed that orally administered Ova induced anti-Ova IgA, IgM, IgG, IgG, and IgG2 antibodies in serum relative to the control piglets gavaged with saline (Pasternak et al., 2015b). These data suggest that the antigen traversed the neonatal gut wall and an adaptive immune response was mounted in the newborn piglet. Further research needs to be performed to establish if an oral vaccine administered when the gut is semi-permeable, prior to gut-closure, can protect against neonatal enteric diseases. Trials should include formulating the vaccine for slow release once it traverses the gut wall to be acted upon by the immune system in the post-neonatal period. Furthermore, there must be con rmation that vaccines administered orally during the neonatal period do not trigger a T regulatory response instead of a stimulatory immune response.

Conclusions
Our research shows that in duodenal, jejunal and ileal IECs, Cy5-Ova does not colocalize with pIgR on the surface of the cells or within pIgR+ endosomes, regardless of whether they were gavaged pre-or postsuckling. Cy5-Ova appears to be largely located within small, medium, and large-sized transport and digestive vacuoles and it was colocalized with RAB7 in the endosomes throughout the small intestine in 0 hour gavaged piglets and the ileum of 1 hour gavaged piglets. Likewise, Cy5-Ova colocalized with lysosomal marker LAMP-1 in the duodenal and jejunal IECs in 0 hour and 1 hour gavaged piglets but only in the ileum in the 0 hour old gavaged piglets. The intake of colostral macromoleucles takes time to process and therefore reduces the uptake and processing of new antigen in the ileum. Whether the ileal uptake of Cy5-Ova in the 1 hr-old gavaged piglets leads to increased transport rather than lysosomal digestion may impact timing of oral vaccine delivery in newborn piglets.

Declarations Ethics Approval and Consent to Participate
This work was approved by the University of Saskatchewan's Animal Research Ethics Board and adhered to the Canadian Council on Animal Care Guidelines for humane animal use.

Availability of Data and Materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.   Background uorescence assessment on pig intestine incubated with secondary antibodies from a control pig not gavaged with Cy5-OVA. These images are representatives of IHC-p performed on duodenum, jejunum, and ileum tissue samples using secondary antibody (A, B, and C) Alexa 555-labeled goat anti-rabbit IgG (H+L) (green), secondary antibody (D, E, and F) anti-mouse IgG2b labeled-FITC (blue), and secondary antibody (G, H and I) anti-mouse IgG1 labeled-FITC (blue). The red colour is background uorescence at 800 nm which corresponds to the uorescence used to detect Cy5-Ova (J, K , and L). All images were taken with a Leica Confocal Microscope at 63X.  pIgR, RAB7 and LAMP-1 detection and Cy5-Ova localization in the jejunum of a 0-hour old gavaged piglets. These images are representatives of IHC-p performed on jejunum tissue samples. Primary antibodies: rabbit anti-pIgR (a,c), mouse anti-RAB7 (d,f), and mouse anti-LAMP-1 (g,i). Secondary antibodies were Alexa 555-labeled goat anti-rabbit IgG (green), anti-mouse IgG2b labeled-FITC (blue), and anti-mouse IgG1 labeled-FITC (blue). Cy5-Ova is shown in red (b,c,e,f,h,i). Apical localization is shown with white arrows. Vacuole, endosome, and/or lysosome localization is shown with white circles. All images were taken with a Leica Confocal Microscope at 63X. pIgR, RAB7 and LAMP-1 detection and Cy5-Ova localization in the ileum of a 0-hour old gavaged piglets.

Consent for Publication
These images are representatives of IHC-p performed on ileum tissue samples. Primary antibodies: rabbit anti-pIgR (a,c), mouse anti-RAB7 (d,f), and mouse anti-LAMP-1 (g,i). Secondary antibodies were Alexa 555-labeled goat anti-rabbit IgG (green), anti-mouse IgG2b labeled-FITC (blue), and anti-mouse IgG1 labeled-FITC (blue). Cy5-Ova is shown in red (b,c,e,f,h,i). Apical localization is shown with white arrows. Vacuole, endosome, and/or lysosome localization is shown with white circles. All images were taken with a Leica Confocal Microscope at 63X. pIgR, RAB7 and LAMP-1 detection and Cy5-Ova localization in the duodenum of a 1-hour old gavaged piglets. These images are representatives of IHC-p performed on duodenum tissue samples. Primary antibodies: rabbit anti-pIgR (a,c), mouse anti-RAB7 (d,f), and mouse anti-LAMP-1 (g,i). Secondary antibodies were Alexa 555-labeled goat anti-rabbit IgG (green), anti-mouse IgG2b labeled-FITC (blue), and anti-mouse IgG1 labeled-FITC (blue). Cy5-Ova is shown in red (b,c,e,f,h,i). Basolateral localization is shown with orange arrows. Lamina propria localization is shown with orange circles. Vacuole, endosome, and/or lysosome localization is shown with white circles. All images were taken with a Leica Confocal Microscope at 63X. pIgR, RAB7 and LAMP-1 detection and Cy5-Ova localization in the jejunum of a 1-hour old gavaged piglets. These images are representatives of IHC-p performed on jejunal tissue samples. Primary antibodies: rabbit anti-pIgR (a,c), mouse anti-RAB7 (d,f), and mouse anti-LAMP-1 (g,i). Secondary antibodies were Alexa 555-labeled goat anti-rabbit IgG (green), anti-mouse IgG2b labeled-FITC (blue), and anti-mouse IgG1 labeled-FITC (blue). Cy5-Ova is shown in red (b,c,e,f,h,i). Apical localization is shown with white arrows. Lamina propria localization is shown with orange circles. Vacuole, endosome, and/or lysosome localization is shown with white circles. All images were taken with a Leica Confocal Microscope at 63X. pIgR, RAB7 and LAMP-1 detection and Cy5-Ova localization in the duodenum of a 0-hour old gavaged piglets. These images are representatives of IHC-p performed on duodenum tissue samples. Primary antibodies: rabbit anti-pIgR (a,c), mouse anti-RAB7 (d,f), and mouse anti-LAMP-1 (g,i). Secondary antibodies were Alexa 555-labeled goat anti-rabbit IgG (green), anti-mouse IgG2b labeled-FITC (blue), and anti-mouse IgG1 labeled-FITC (blue). Cy5-Ova is shown in red (b,c,e,f,h,i). Apical localization is shown with white arrows. Localization between IECs is shown with purple arrows. Vacuole, endosome, and/or lysosome localization is shown with white circles. All images were taken with a Leica Confocal Microscope at 63X. Quanti cation of uorescence for anti-pIgR, RAB7, LAMP-1, and Cy5-OVA in piglets gavaged with Cy5-OVA 0 or 1 hour after birth. Immunohistochemistry was performed on Duodenum (A), Jejunum (B) and Ileum (C) from control, 0 hour gavaged and 1 hour gavaged piglets using rabbit anti-pIgR, mouse anti-RAB7, and mouse anti-LAMP-1 primary antibodies and Alexa 555-labeled goat anti-rabbit IgG(green), antimouse IgG2b labeled-FITC (blue), and anti-mouse IgG1 labeled-FITC (blue) secondary antibodies,