Comparison of the Small Intestine of 0-day-old Neonatal Piglets vs. 21-day-old Weaned Piglets


 Background: Neonatal piglets are susceptible to intestinal infections . Gut is the body’s major immune structure and the intestinal mucosa, which is composed of intestinal epithelial cells (IELs) and subepithelial natural immune cells, is considered as the primary site for eliciting local immune responses to foreign antigens. This study compared the intestinal immune cells of neonatal and weaned piglets to provide a theoretical and mechanistic basis for preventing intestinal infectious diseases. Results: Histological analyses of weaned piglet intestines showed increased crypt depth, high IEL count, and increased areas of ileal Peyer’s patches. Additionally, the duodenal and ileal villi of weaned piglets were longer than those of neonatal piglets. Expression of claudin-3 protein in weaned piglets was remarkably high as compared with neonatal piglets. The number of CD3 + T cells, goblet cells, and secretory cells was high in the small intestines of weaned piglets in vivo. Contrarily, secretory IgA-positive cell numbers in the jejunum remained unchanged between neonatal and weaned piglets. Gene expression of 12 pattern recognition receptor (PRR) (TLR1–10, MDA5, and RIG-I) was examined in neonatal and weaned piglet small intestine (duodenum, jejunum , and ileum). The pattern of mRNA expression level of most PRR genes in the duodenum and jejunum was inverse of that in the ileum. Compared with weaned piglets, there were significantly fewer intestinal lymphocytes at birth in neonatal pigs. Conclusions: The physical, biochemical, and immune-related components of neonatal and weaned piglet small intestines were investigated to provide preliminary data on the pathogenetic mechanism for future studies.

microorganisms [3,4]. In addition, gut is the major immune organ of the body and the intestinal mucosa is thought to be the primary site for eliciting and mediating local immune responses [3,5].
The anatomy and physiology of porcine digestive system are highly comparable to those of humans, which favors the use of pigs as a suitable animal model, closely resembling humans. Therefore, pigs are considered to be an ideal animal model to investigate human intestinal diseases and to understand the biological pathways underlying mucosal functions and development [6,7]. Infectious diseases of the digestive tract are the most frequent and recurrent conditions in swine industry.
Infections due to E. coli, porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus, and transmissible gastroenteritis coronavirus can suppress the feed conversion efficiency and represent major threats to the swine industry [8][9][10].
Changes in the intestinal morphology and structure of piglets mainly occur at birth and during weaning [11]. The porcine intestinal immune system is immature at birth [12], and thus, most pathogenic microorganisms mainly infect neonatal piglets. Normally weaned pigs are resistant to infection by pathogenic microorganisms. Many studies have reported on the development of intestinal structures of pig small intestine [13]. However, limited studies have compared the intestinal structures of neonatal and weaned pigs. The intestinal mucosal barrier has physical, biochemical, and immunological components [14]. In the present study, we compared the physical, biochemical, and immunological components of neonatal and weaned piglet intestines to study the mechanism of disease pathogenesis and to provide preliminary data for further studies in future.

Methods Animals
A total number of six, three (21-days-old; weighting, 810 kg) and three (0-day-old; weighting, 1.101.30 kg) male crossbred Duroc/Landrace/Yorkshire piglets were obtained from Jiangsu Huai'an Pig Farm (Huai'an, China). The piglets were kept in Jiangsu Huai'an Pig Farm with a constant humidity of 60% and 26˚C temperature at light/dark cycle after each 12 h with free access to water and food.

Material Collection
At the day of euthanasia, each piglet was sacrificed by anesthetizing with an intravenous injection of pentobarbital sodium (100 mg/kg) before sample collection. To ensure whether the piglets were completely anesthetized, the eyelids and withdrawal reflex were checked before opening the thorax.
Duodenum, jejunum, and ileum tissues samples were immediately collected from the piglets. All procedures performed on the animals were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University and followed the National Institutes of Health guidelines for the performance of animal experiments.

Histological analysis
The tissue samples of small intestine (duodenum, jejunum, and ileum) were fixed in 4% paraformaldehyde for 48 h at room temperature. After fixation, the samples were sectioned in small pieces to fit the glass slides and then dehydrated in a graded alcohol series (75, 85, 95, 100, and 100% ethanol, each for 1 min). The dehydrated blocks were embedded in paraffin wax and keep in room temperature to dry completely. The tissue samples were sectioned longitudinally on glass slide at 5-μm-thick by using microtome. The sections were dried horizontally on a warming tray overnight at 37 °C, dewaxed in xylene, and rehydrated in a graded series of ethanol (100, 90, 80 and 70%, each for 1 min), and washed in PBS for hematoxylin and eosin (H&E) staining. The sections stained with H&E were examine by light microscopy (BH-2, Olympus). Villus height and crypt depth were measured (single-blind) by an observer using computer-assisted morphometry (Image-Pro Plus software). The area of lymphoid follicles in ileal Peyer's patches (PPs) was also measured.

Immunohistochemistry
After deparaffinization and rehydration, the sections were subsequently poached into a citrate buffer (pH 6) at 90-95˚C for 15 min to retrieve antigens. Then, the sections were treated with 0.3% hydrogen peroxide at room temperature for 15 min to quench endogenous peroxidase and wash with PBS. To avoid the non-specific binding of antibodies the sections were blocked with 5% bovine serum albumin for 30 min at room temperature. After incubating with the primary antibodies overnight at 4˚C, sections were treated with biotinylated secondary antibodies for 1 h at room temperature. To visualize the positive cells, sections were treatment with diaminobenzidine (DAB) for 60 min at room temperature, and then sealed with neutral balata. The respective isotypes were used as negative controls. The sections were visualized by light microscope (Olympus CX23; Olympus Corporation, 5 Tokyo, Japan) at a magnification of x400 or x100. Different fields of each tissue in each piglet were counted for the statistical analysis.

Immunofluorescence staining
The tissue sections were incubated with 0.4% Triton X-100 in PBS for 5 min. After blocking with 5% bovine serum albumin in PBS for 1 h, the sections were stained with UEA-I antibodies at room temperature for 2 h. PBS was used instead of antibody in control samples. After staining with DAPI, the sections were observed under a confocal laser microscope (LSM-710; Zeiss, Oberkochen, Germany) visualized by CLSM (LSM 710, Zeiss, Oberkochen, Germany).

RNA isolation and RTqPCR.
Total RNA was extracted from duodenum, jejunum, and ileum tissues using a TRIzol™ Plus RNA Purification kit according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.). 1 ug of total purified RNA was reverse transcribed to cDNA by using PrimeScript™ RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. Reverse transcription was performed at one cycle of 37˚C for 15 min followed by 85˚C for 5 sec. A total of 2 μl diluted cDNA (vol:vol, 1:5) was used to perform RT-qPCR analysis, by ABI 7500 PCR system (Life Technologies; Thermo Fisher Scientific, Inc.) using SYBR-Green qPCR Master Mix (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocol. The thermo cycler reaction involved a pre-incubation of 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec and 60˚C for 31 sec. Data were normalized against GAPDH mRNA level and are expressed as fold differences between control and treated cells calculated using the 2 -ΔΔCT method. Results were expressed as means ± standard deviation (SD). Analysis of variance and unpaired Student's t-tests were employed to determine statistical significance differences among multiple groups. The differences were considered significant at *P < 0.05, **P < 0.01.

Histological characteristics of neonatal and weaned piglet intestines
We compared the differences in the development of porcine gut between neonatal and weaned piglets. The duodenum, jejunum, and ileum of the small intestine of neonatal and weaned piglets were sampled and stained using hematoxylin-eosin. As shown in Fig. 1a, microvilli in the small intestine of neonatal piglets appeared different from those of weaned piglets. Weaned piglets had marked shortening, clubbing, and blunting of the duodenal villi, whereas the ileal villi of weaned piglets were longer than those of the neonatal piglets. Moreover, the jejunal villus length between neonatal and weaned piglets showed no difference. Additionally, both the crypt depth (Fig. 1b) and number of IELs (Fig. 1c) were markedly high in weaned piglets. To identify the differences in the intestinal immune system, we compared the size of ileal Peyer's patches (PPs) in the neonatal and weaned piglets (Fig. 1d). The weaned piglets had significantly larger and more mature PPs than those in the neonatal piglets; moreover, the boundaries between the partial PPs were obscure in neonatal piglets.
Weaned piglets have superior intestinal barrier function than neonatal piglets Tight junctions are the important determinants of epithelial barrier functions [15]. Consequently, we compared the expression of genes coding for tight junction proteins in various areas of the small intestine between the neonatal and weaned piglets. As shown in Fig. 2a, compared with that in the neonatal piglets, the relative expression of claudin and occludin transcripts in the intestinal mucosa was remarkably high in weaned piglets; however, the expression of E-cadherin and ZO-1 transcripts showed no significant change. We performed immunohistochemical staining of small intestinal tissue sections with anti-claudin-3 antibodies to further compare the difference in the expression of tight junction proteins in various gut areas between the neonatal and weaned piglets. In weaned piglets, the expression of claudin-3 proteins was remarkably high with that in neonatal piglets (Fig. 2b).
Characteristics of mucosal immunity in neonatal and weaned piglet intestines 7 CD3 + T lymphocytes represent the major T lymphocyte subtype; therefore, the number of CD3 + T lymphocytes could represent the absolute T lymphocyte count. We compared the number of CD3 + T lymphocytes in the small intestine between neonatal and weaned piglets. The CD3 + T lymphocyte distribution patterns were examined by immunohistochemistry. Immuno-positive cells were stained brown. Weaned piglets have more CD3 + T lymphocytes in the duodenum, jejunum, and ileum of the small intestine than those of neonatal piglets (Fig. 3a). SIgA is the main immunoglobulin isotype in animals and is primarily secreted across the intestinal mucosal surface, especially in the small intestine. SIgA plays an important role in intestinal mucosal immunity and is an index of intestinal mucosal immunity. The number of IgA + B-cells on the small intestine mucosal surface were examined by immunohistochemistry. Immuno-positive cells were stained brown. The results indicated that weaned piglets have more IgA + B-cells in the duodenum and ileum of the small intestine than those of neonatal piglets, although there was no significant difference in the jejunal villus length between the neonatal and weaned piglets (Fig. 3b). Goblet cells (GCs) are specialized epithelial cells that secrete anti-microbial proteins, chemokines, and cytokines, and have important innate immunity-related functions besides intestinal barrier maintenance. PAS-stained intestinal sections showed the presence of GCs in the intestinal mucosa surface epithelia of neonatal and weaned piglets. These cells, which appeared blue after PAS staining, were typically circular and cup-shaped and were located in the intestinal lamina propria. As shown in Fig. 3c, the weaned piglets have more GCs in the duodenum, jejunum, and ileum of the small intestine than those of neonatal piglets. UEA-1, a universal marker of secretory cells, was also examined to determine the activation status of the intestinal cells. The results showed that weaned piglets had a higher proportion of intestinal secretory cells as compared with neonatal piglets (Fig. 3d).
Intestinal mucosa of neonatal and weaned piglets show differential modulation on PRRs genes PRRs in the intestinal mucosa, such us the toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), are important sensors in host-microorganism crosstalk. These receptors are expressed on various mucosal cell types. PRRs play a critical role in the detection of microorganisms and in the elicitation of 8 inflammatory and immune responses. Expression of 12 PRR genes (TLR1-10, MDA5, and RIG-I) was first examined along the small intestine (duodenum, jejunum, and ileum) in both neonatal and weaned piglets. The weaned piglets showed a significant increase in the expression of TLR3, TLR5, MDA5, and RIG-I genes in the duodenum as compared to neonatal piglets. In contrast, duodenal TLR6 expression was downregulated in weaned piglets as compared with neonatal piglets (Fig. 4a). In addition, on assessing the expression of multiple genes, the expression of most of the tested genes TLR-1, 2, 4, 5, 10, and MDA5 (Fig. 4b) were higher in jejunum of weaned piglets than those of neonatal piglets. In contrast, the expression of few genes such as TLR-3, 5, 6, 7, MDA5, and RIG-I in the ileum of weaned piglets were lower than those of neonatal piglets (Fig. 4c).
Percentage of CD3+, CD4+, and CD8+ T lymphocytes in peripheral blood mononuclear cells Lymphocytes are a major component of the peripheral innate immune system and are responsible for engulfing and killing pathogens during an infection [16]. Neonatal lymphocytes have quantitative deficiencies. As shown in Fig. 5, the number of CD3 + cells in the blood of neonatal and weaned piglets was 16.1% and 39.9%, respectively, while the number of CD4 + and CD8 + cells in the blood of neonatal and weaned piglets was 2.69% and 7.67%, respectively. Therefore, the number of lymphocytes were significantly fewer at birth as compared to that in adults, which is maintained at physiological levels. These results suggest that neonatal piglets are more susceptible to contracting infection.

Discussion
Various enteric pathogens can cause intestinal diseases in piglets or adult pigs and lead to tremendous economic losses [10]. The small intestine is the primary absorbing organ and is divided into three parts-duodenum, jejunum, and ileum. The jejunum absorbs most of the nutrients, and the bile salt is absorbed at the end of the ileum [4]. Our study compared the morphology, immune cell composition, as well as the expression of tight junction proteins and receptors in the small intestine in 0-day-old neonatal and 21-day-old weaned pigs to investigate age-dependent severity of disease, lesions, and deaths.
The small intestine has numerous villi that increase the internal surface area to improve the efficiency 9 of nutrient absorption. We found that the length of the duodenal villi of neonatal piglets was significantly longer than that of weaned pigs. However, a contrasting observation was noted in the ileum (Fig. 1a). This might have been due to different dietary patterns. Neonatal piglets only consume milk, whereas weaning pigs are provided with solid feed.
The digestion and absorption of feed requires a lot of bile salt. Moreover, the food passing through the intestine could injury the internal cells; however, the intestinal crypt has a large number of stem cells which maintain the intestinal epithelium integrity [17,18]. Therefore, the crypt depth of weaned piglets was significantly higher than that of neonatal piglets (Fig. 1b) possibly to produce more stem cells. There are multiple antigens in the gastrointestinal tract. Lymphocytes protect the body from pathogens. Villagómez et al. proved that the IELs act as sentinels for maintaining the mucosal barrier integrity [19]. Piglets mainly rely on maternal antibodies to resist pathogenic invasion [20].
Consequently, the number of IELs in each villus (Fig. 1c) and PPs (Fig. 1d) of weaning piglets were markedly higher than that of neonatal piglets. In addition, compared with weaned piglets, the immune system is not fully developed in neonatal piglets because of which there are lesser numbers of immune cells (Fig. 2) such as CD3 + T cells, IgA + cells, GCs, secretory cells, etc. in the peripheral circulation.
Tight junction is a cell adhesion apparatus, a barrier and/or channel, in the space among adjacent epithelial cells [21]. First, we examined the expression genes coding for major intestinal tight junction proteins in neonatal and weaned piglet small intestinal tissue (Fig. 3a). We found no significant difference in ZO-1 and E-cadherin gene expression. However, the expression of claudin and occludin genes were different in the jejunum, and moreover, claudin-3 expression showed obvious dissimilarity at the protein level. This might have been due to selective gene expression. These results prove that weaned pigs have better intestinal epithelial integrity than neonatal piglets.
PRRs are membrane-bound receptors either expressed on the cell surfaces or associated with intracellular vesicles that specifically bind to pathogen-associated molecular patterns (PAMPs), homologous proteins shared among various microorganisms [22,23]. RNA viruses, including PEDV, can interact with a large number of PRRs in the intestinal mucosa, such as TLRs and RLRs [24]. This interaction plays a critical role in the activation of the innate immune response. When microbes breach physical barriers such as the skin and mucosa, TLR recognizes their PAMPs and activate the immune responses [25]. We found that the number of TLR in the duodenum and jejunum of weaning piglets was relatively higher than that of neonatal piglets; however, the ileum showed contrasting results for TLR expression. Only few cases of ileal diseases have been reported. The innate immune system consists of granulocytes (mainly neutrophils), antigen presenting cells, natural killer cells, and γδ-T cells [26]. Lymphocytes have the properties of mature antigenexperienced cells and contribute to adaptive immune response activation against the invading pathogens. As shown in Fig. 5, the number of lymphocytes were significantly fewer in 0-day-old piglets as compared to that in adult pigs with physiological levels of lymphocytes. Therefore, these results suggest that neonatal piglets are more susceptible to diseases following pathogenic invasion.

Conclusion
For the first time we evaluated the differences in the small intestine of neonatal and weaned piglets.
Our findings indicate that neonatal piglets are more susceptible to pathogenic microorganisms and provide preliminary data on the pathogenetic mechanism for future studies.   immunofluorescent staining of secretory cell in pig small intestine. Positive cells were quantified by densitometry analysis, and were calculated in 10 random fields. Scale bars: 100 μm. Differences were considered significant at (*) 0.01 < p < 0.05, (**) p < 0.01. 19 20 Figure 4 Expression of PRRs genes mRNA expression in pig small intestine between neonatal and weaned piglets. a-c . The mRNA levels of TLR1, TLR2, TLR 3, TLR4, TLR5, TLR6, TLR7, TLR8  ARRIVEguidelines.rtf