Advertisement

Diet- and Colonization-Dependent Intestinal Dysfunction Predisposes to Necrotizing Enterocolitis in Preterm Pigs

Published:February 20, 2006DOI:https://doi.org/10.1053/j.gastro.2006.02.026
      Background & Aims: Preterm birth and formula feeding are key risk factors associated with necrotizing enterocolitis (NEC) in infants, but little is known about intestinal conditions that predispose to disease. Thus, structural, functional, and microbiologic indices were used to investigate the etiology of spontaneous NEC development in preterm pigs. Methods: Piglets were delivered by cesarean section at 92% gestation, reared in infant incubators, and fed infant formula or colostrum every 3 hours (n = 120) until tissue collection at 1–2 days of age. Results: Clinical and histopathologic signs of NEC were observed in 57% of pigs fed FORMULA (26/46) and in 5% of pigs fed COLOSTRUM (2/38) (P < .05). Relative to COLOSTRUM, both healthy and sick FORMULA pigs had reduced intestinal villous heights, enzyme activities, nutrient absorption, and antioxidant levels and higher inducible nitric oxide synthetase activity (P < .05). In healthy pigs, mucosal microbial diversity remained low and diet independent. NEC pigs showed bacterial overgrowth, and a high mucosal density of Clostridium perfringens was detected in some but not all pigs. Germ-free conditions and antiserum against Clostridium perfringens toxin prevented intestinal dysfunction and NEC in formula-fed pigs, whereas the gut trophic factors, epidermal growth factor, and glucagon-like peptide 2 had limited effects. Conclusions: A subclinical, formula-induced mucosal atrophy and dysfunction predispose to NEC and bacterial overgrowth. The adverse feeding effects are colonization dependent and may be reduced by factors in colostrum that include antibodies against aggressive toxins such as those of Clostridium perfringens.

      Abbreviations used in this paper:

      BSA (bovine serum albumin), CP (Clostridium perfringens), EGF (epidermal growth factor), GLP-2 (glucagon-like peptide 2), GIT (gastrointestinal tract), HSA (human serum albumin), IgG (immunoglobulin G), NEC (necrotizing enterocolitis), NOS (nitric oxide synthetase), PCR (polymerase chain reaction), SGLT-1 (sodium-coupled glucose transporter 1), T-RFLP (terminal restriction fragment length polymorphism)
      Infants delivered preterm have an immature gastrointestinal tract (GIT) with underdeveloped capacity to digest and absorb carbohydrates and other nutrients. When the nutrient load exceeds the digestive capacity, bacterial overgrowth, and fermentation may occur and predispose to the most serious GIT disease in neonatal wards, necrotizing enterocolitis (NEC).
      • Shulman R.J.
      • Schandler R.J.
      • Lau C.
      • Heitkemper M.
      • Ou C.N.
      • Smith E.O.
      Early feeding, feeding tolerance and lactase activity in preterm infants.
      • Berseth C.L.
      • Bisquera J.A.
      • Paje V.U.
      Prolonging small feeding volumes early in life decreases the incidence of necrotizing enterocolitis in very low birth weight infants.
      • Caicedo R.A.
      • Schanler R.J.
      • Li N.
      • Neu J.
      The developing intestinal ecosystem implications for the neonate.
      Feeding formula is associated with a higher incidence of NEC than mother’s milk, but it is not known how the absence of natural milk, combined with the consequences of preterm birth (hypoxia, hyperthermia, GIT immaturity), may predispose to NEC. The symptoms can be induced in newborn rodents and pigs by installation of acidified diets into the GIT lumen, coupled with hypothermia, and/or hypoxia exposure.
      • Caplan M.S.
      • Jilling T.
      The role of polyunsaturated fatty acid supplementation in intestinal inflammation and neonatal necrotizing enterocolitis.
      • Di Lorenzo M.
      • Krantis A.
      Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.
      Such acute disease models have provided important information about some pathologic mechanisms, but they do not provide insights into intestinal physiology prior to spontaneous disease outbreak.
      NEC is a disease that typically starts with feeding intolerance, reduced bowel motility, and moderate fecal blood loss. As NEC progresses, there is intestinal dilation and immotility and gas accumulation in the intestinal lymph vessels and portal vein (pneumatosis intestinalis). Finally, peritonitis and transmural necrosis may develop, and the affected bowel segment becomes perforated (Bell stages I, II, and III).
      • Walsh M.C.
      • Kliegman R.M.
      Necrotizing enterocolitis treatment based on staging criteria.
      • Bell M.J.
      • Ternberg J.L.
      • Feigin R.D.
      • Keating J.P.
      • Marshall R.
      • Barton L.
      • Brotherton T.
      Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging.
      Although the ischemic affliction is usually restricted to the ileum and proximal colon, necrosis is sometimes found throughout the GIT, including the stomach.
      • Walsh M.C.
      • Kliegman R.M.
      Necrotizing enterocolitis treatment based on staging criteria.
      • Travadi J.N.
      • Patole S.K.
      • Simmer K.
      Gastric pneumatosis in neonates revisited.
      The incidence and severity of NEC are highly variable, and subclinical intestinal dysfunctions may be present in children who appear clinically healthy.
      Inappropriate immunologic responses of premature enterocytes to bacterial colonization have been implicated in the development of NEC.
      • Nanthakumar N.N.
      • Fusunyan R.D.
      • Sanderson I.
      • Walker W.A.
      Inflammation in the developing human intestine a possible pathophysiologic contribution to necrotizing enterocolitis.
      • Claud E.C.
      • Walker W.A.
      Hypothesis inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis.
      Although the Clostridium species have been frequently associated with NEC,
      • De La Cochetiere M.F.
      • Piloquet H.
      • Des Robert C.
      • Darmaun D.
      • Galmiche J.P.
      • Roze J.C.
      Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants the putative role of clostridium.
      • Waligora-Dupriet A.J.
      • Dugay A.
      • Auzeil N.
      • Huerre M.
      • Butel M.J.
      Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model.
      no single pathogen is causative, and the ability of the microflora to colonize the epithelium and to ferment unabsorbed nutrients may be more important than the strain itself. Typically, NEC patients have higher circulating levels of proinflammatory cytokines (eg, tumor necrosis factor α [TNF-α], interleukin [IL]-1β, IL-6) but not until the inflammatory responses have resulted in severe necrosis.
      • Morecroft J.A.
      • Spitz L.
      • Hamilton P.A.
      • Holmes S.J.
      Plasma interleukin-6 and tumour necrosis factor levels as predictors of disease severity and outcome in necrotizing enterocolitis.
      • Edelson M.B.
      • Bagwell C.E.
      • Rozycki H.J.
      Circulating pro- and counterinflammatory cytokine levels and severity in necrotizing enterocolitis.
      Thus, the surge in plasma inflammatory mediators appears to be a relatively late phenomenon during disease progression. Poorly coordinated production of nitric oxide (NO) and NO synthetase (NOS) isoforms during the early phases of disease could result in the altered intestinal blood flow and ischemic damage typically seen in NEC.
      • Di Lorenzo M.
      • Krantis A.
      Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.
      Collectively, these findings suggest that the early phases of NEC include a combination of inadequate digestion and nutrient fermentation, localized ischemia, inappropriate immune responses induced by resident bacteria, and decreased mucosal protection (eg, antioxidants
      • Finer N.N.
      • Peters K.L.
      • Hayek Z.
      • Merkel C.L.
      Vitamin E and necrotizing enterocolitis.
      ). However, neither of the proposed functional GIT deficits has been demonstrated in a mammalian animal model that incorporates prematurity, diet, bacterial colonization, and spontaneous outbreak of disease.
      We hypothesized that NEC would develop spontaneously in a significant proportion of preterm, FORMULA pigs and that FORMULA-induced deficits in GIT function would be colonization-dependent and present in clinically healthy tissue prior to pathologic manifestations of NEC. Hence, the first objective (experiment 1) was to document spontaneous development of NEC in FORMULA pigs and to identify functional deficits in nonnecrotic tissues from FORMULA pigs. The second objective was to test whether a FORMULA-induced mucosal atrophy and dysfunction in preterm pigs would be colonization-dependent, using gnotobiotic experimental conditions (experiment 2). Finally, a third objective was to test the potential protective effects of providing antiserum against some common pathogens in neonatal piglets (Clostridium perfringens, Escherichia coli)
      • Feng J.
      • El-Assal O.N.
      • Besner G.E.
      Heparin-binding EGF-like growth factor (HB-EGF) and necrotizing enterocolitis.
      or administration of 2 gut trophic factors, epidermal growth factor (EGF) or glucagon-like peptide 2 (GLP-2) (experiment 3). EGF was chosen because of its presence in maternal milk, the expression of EGF receptors by the preterm GIT, and the reported potential for EGF to reduce NEC incidence.
      • Dvorak B.
      Epidermal growth factor and necrotizing enterocolitis.
      The choice of GLP-2 was based on its trophic and functional effects on the immature mucosa
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Sangild P.T.
      Glucagon-like peptide 2 enhances maltase-glucoamylase and sucrase-isomaltase gene expression and activity in parenterally-fed premature neonatal pigs.
      and the stimulation of intestinal blood flow and metabolism in newborn pigs.
      • Guan X.
      • Stoll B.
      • Lu X.
      • Tappenden K.A.
      • Holst J.J.
      • Hartmann B.
      • Burrin D.G.
      GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets.

      Materials and Methods

       Experiment 1: Structural and Functional Indices of NEC Development

      Forty-five pigs (Danish Landrace × Large White) from 6 sows were delivered by cesarean section at 105–108 days gestation (term = 115 days), as described in detail previously.
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      Pigs from each litter were randomly allocated to receive either milk replacer (FORMULA, n = 14), sow’s colostrum (COLOSTRUM, n = 13), or were killed (sodium pentobarbitone, 60 mg kg-1, intravenously, [IV]) immediately after birth (NEWBORN, n = 18). Pigs assigned to the feeding groups were transferred to heated infant incubators within 5 minutes of birth and supplied with extra oxygen over the first 12 hours of life until arterial blood oxygenation exceeded 95%. Within 4 hours of delivery, each pig was fitted with a vascular catheter (infant feeding tube, 4F; Portex, Kent, United Kingdom) inserted into the dorsal aorta via the transected umbilical cord and prepared with an orogastric feeding tube (6F, Portex).
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      The pigs were fed COLOSTRUM or FORMULA (15 mL/kg) every 3 hours after placement of the catheters. The COLOSTRUM group received passive immunologic protection via absorption of colostral immunoglobulins. The FORMULA pigs were immunized systemically by arterial administration of serum taken from their own mothers that had received no vaccination regime (15 mL/kg over the first 12 hours). This provides adequate immunologic protection of preterm, COLOSTRUM-deprived newborn pigs.
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      Before tissue collection at 20–30 hours after birth (mean age, 24 ± 2 hours), an arterial blood sample was taken, and the pigs were killed with an arterial injection of pentobarbitone (60 mg/kg, IV). Blood was analyzed immediately (blood chemistry values), or the plasma was separated and stored (−20°C) for later biochemical measurements. Tissue samples were collected (see below, Tissue Collection and Blood Chemistry section) and stored fixed in formaldehyde or frozen in liquid nitrogen for later structural, biochemical, and microbiologic analyses.

       Experiment 2: Gnotobiotic Preterm Pigs

      A total of 35 cesarean-delivered pigs from 3 litters (Danish Landrace × Large White, 106 days gestation) were randomly allocated to 3 treatment groups: COLOSTRUM (COLOSTRUM, n = 11), FORMULA (FORMULA, n = 12), and FORMULA pigs delivered and reared sterile (FORMULA/GNOTO, n = 12). Sterile delivery and transfer via an iodine bath into a gnotobiotic incubator followed standard procedures. Feeding regimen and tissue collection procedures at 24–40 hours were as described for experiment 1, except that samples of nasal fluid and colon contents were taken prior to tissue collection, for anaerobic and aerobic microbiology. Analyses of tissues from experiment 2 focused on the clinical and histopathologic signs of NEC in the 3 treatment groups combined with some selected structural and functional analyses (villous morphology, enzyme activities).

       Experiment 3: Administration of EGF, GLP-2, or Bacterial Antiserum

      A total of 58 cesarean-delivered pigs from 5 litters (Danish Landrace × Large White, 105–108 days gestation) were allocated to 5 treatment groups. Four of the groups received either sow’s COLOSTRUM (COLOSTRUM, n = 14), formula (FORMULA, n = 20), FORMULA + EGF (human EGF20–31, Bachem H-6175, 20 μg/kg, given intraperitoneally [IP] every 6 hours, n = 9, FORMULA/EGF), or FORMULA + GLP-2 (human GLP-21–33, NOVO Nordisk, Bagsværd, Denmark, 50 μg/kg, given IP every 6 hours, n = 9, FORMULA/GLP-2). The dose of EGF was chosen based on a dose that has previously been shown to induce marked mucosal maturation in newborn pigs.
      • James P.S.
      • Smith M.W.
      • Tivey D.R.
      • Wilson T.J.
      Epidermal growth factor selectively increases maltase and sucrase activities in neonatal piglet intestine.
      The GLP-2 dose was chosen based on the observation that a similar dose administered over several days to preterm parenterally fed pigs induced marked intestinal mucosal growth.
      • Petersen Y.M.
      • Hartmann B.
      • Holst J.J.
      • Le Huerou-Luron I.
      • Bjornvad C.R.
      • Sangild P.T.
      Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development.
      While the FORMULA, FORMULA/EGF, and FORMULA/GLP-2 groups received normal sow’s plasma for passive immunization, the remaining FORMULA/ANTI group received plasma from a sow immunized with a commercially available vaccine (Toxicol; Schering-Plough, Farum, Denmark) directed against the α, β, and ϵ toxins of Clostridium perfringens (CP) and Escherichia coli F4ab, F4ac, F5, and F6 antigens (15 mL/kg, IV, within 12 hours postpartum, n = 6). Vaccination of pregnant sows with this product normally provides full protection of newborn piglets against necrotizing enteritis related to CP α, β, and ϵ toxins via their uptake of antibodies from colostrum ingestion.
      • Springer S.
      • Selbitz H.J.
      The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.
      The circulating antibody titer for CP β toxin (international units [IU] per mL) was measured in plasma by standard ELISA techniques using purified CP β toxin obtained from pure cultures of CP type C.
      • Springer S.
      • Selbitz H.J.
      The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.
      The sow’s serum was provided to FORMULA piglets systemically because FORMULA is known to facilitate poor uptake of intestinal immunoglobulins.
      • Jensen A.R.
      • Elnif J.
      • Burrin D.G.
      • Sangild P.T.
      Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
      Delivery of pigs, feeding, and sample collection procedures were identical to those in experiment 1, except that all the pigs were reared for a slightly longer period (40 ± 4 hours). Analyses of tissues from experiment 3 focused on the clinical and histopathologic signs of NEC in the 5 treatment groups combined with some selected structural and functional analyses (villous morphology, enzyme activities). Finally, distal small intestine samples were used for molecular microbiology analyses (terminal restriction fragment length polymorphism [T-RFLP] and fluorescent in situ hybridization).

       Diets

      Colostrum was pooled from different sows that were milked within 6 hours of parturition and was kept frozen until use and contained 6800 kJ/L, 146 g protein per L, and other nutrients as described previously.
      • Jensen A.R.
      • Elnif J.
      • Burrin D.G.
      • Sangild P.T.
      Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
      All COLOSTRUM was collected from sows that had been vaccinated prior to delivery (90 days gestation) with the product mentioned above (Toxicol). The milk formula was prepared by combining 3 commercial products (SHS International, Liverpool, UK) used for feeding hospitalized infants at 0–2 years of age (Table 1). The principal source of carbohydrate was hydrolyzed corn syrup (maltodextrins, Pepdite), the fat was largely obtained from coconut oil (MCT Liquigen), and the protein was provided by a mixture of low molecular proteins from cow’s milk whey and vegetables (Pepdite + Maxipro). The formula was mixed to have energy and protein levels comparable with sow’s milk.
      • Jensen A.R.
      • Elnif J.
      • Burrin D.G.
      • Sangild P.T.
      Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
      • Sangild P.T.
      • Xu R.J.
      Colostrum.
      The osmolality of the formula diet was 182 mOsm/L, compared with 344 mOsm/L for colostrum.
      Table 1The Nutrient Composition of Formula (per L formula)
      Macronutrients and vitaminsMinerals (mg)Amino acids (g)
      Energy (kJ)4151Na345Ala3.1
      Protein (g)63.9K728Arg1.9
      Carbohydrate (g)46.9Cl245Asp6.0
      Total sugars (g)9.0Ca513Cys1.9
      Lactose (g)5.2P392Glu1.4
      Fat (g)61.3Mg66Gln8.5
      Saturated fat (g)44.3Fe5.6Gly2.1
      Monounsaturated fat (g)9.7Cu304His1.3
      Polyunsaturated fat (g)3.7Zn4.0Ile3.9
      Vitamin A (μg)422Mn0.30Leu6.2
      Vitamin D (μg)6.8I0.038Lys5.2
      Vitamin E (mg)2.6Mb0.011Met1.7
      Vitamin C (mg)32Se0.009Phe3.0
      Vitamin K (μg)17Cr0.008Pro3.8
      Vitamin B1 (mg)0.3Ser2.8
      Vitamin B2 (mg)0.5Thr4.0
      Vitamin B6 (mg)0.4Trp1.5
      Vitamin B12 (μg)1.0Tyr2.7
      Folacin (μg)30Val4.1
      Biotin (μg)21Tau0.03
      NOTE. Data are calculated from prescribed contents in the commercially available products (80 g Peptide, 70 g Super Soluble Maxipro, 75 mL MCT Liquigen per 1000 mL formula, all products kindly donated by SHS International, Liverpool, UK).

       NEC Evaluation

      The pigs were observed at least every 3 hours, with the onset of NEC based on lethargy, abdominal distention, and bloody diarrhea. Each animal was subsequently evaluated using a combination of the clinical symptoms just before death, and the magnitude of GIT lesions from macroscopic and histologic examination of tissues from 4 regions: stomach, proximal and distal small intestine, and proximal colon. Pigs without clinical signs of discomfort and feeding intolerance, and lacking abdominal distention or macroscopic hemorrhage, edema, or bleeding of the gastrointestinal mucosa, and with no mucosal abnormality in any of the 4 regions, were assigned a grade 1. Grades 2–6 were assigned to pigs exhibiting feeding intolerance, runny stool with or without blood, abdominal distention with or without intestinal pneumatosis. The specific score was determined by the average degree of mucosal alteration and severity of lesions in the 4 GIT regions. Specifically, when 20% or more of the mucosa in a particular region was hyperemic with clearly stunted villi, it was assigned a grade of 2. Regions graded as 3 and 4 were characterized by more widespread hemorrhagic and partly necrotic mucosa, and grades 5 and 6 were assigned when the damage was extensive and widespread, with evidence of transmural necrosis. Pigs were considered to have clinical signs of NEC when the average score for the 4 regions exceeded 1.5. Thus, the minimum required for NEC diagnosis was extensive mucosal swelling and hyperemia in at least 1 of the 4 GIT regions. Digital photos were taken of the GIT harvested from all pigs for later confirmation of the data collected at necropsy.

       In Vivo Absorption of Macromolecules and Galactose From Oral Test Solutions

      During the first 1–2 postnatal days, enterocytes of the term pig intestine absorb intact protein macromolecules, including colostral immunoglobulins.
      • Jensen A.R.
      • Elnif J.
      • Burrin D.G.
      • Sangild P.T.
      Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
      Transcytosis was evaluated in vivo before clinical signs of NEC and in vitro after tissue harvest. Bovine serum albumin and human serum albumin (BSA; A-4503, or HSA; A-1653, respectively; Sigma Chemical Co, St. Louis, MO) were added to the food (colostrum or formula, 20 mg/mL) at the first feeding (0 hours, BSA) or at 12 hours (HSA). Arterial blood samples were taken at intervals (0, 3, 6, 9, and 12 hours postfeeding) for measurement of circulating BSA and HSA levels. Plasma concentrations of BSA, HSA, and porcine immunoglobulin G (IgG) were measured by quantitative immunoelectrophoresis.
      • Jensen A.R.
      • Elnif J.
      • Burrin D.G.
      • Sangild P.T.
      Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
      The absorption of galactose following an oral load of lactose was investigated at 18–24 hours after birth and before any pigs exhibited clinical symptoms of NEC. Lactose was dissolved in sterile water (10%) and was administered via the orogastric tube at a dose of 1.5 g/kg body weight 3 hours after the last feeding. Arterial blood samples were collected before and at 10, 20, 30, 40, and 70 minutes after the lactose bolus. Plasma galactose was measured by spectrophotometry using galactose-dehydrogenase (Boehringer Mannheim, Darmstadt, Germany). An increase in plasma galactose levels following an oral load of lactose reflects the combined activity of brush border lactase-phloridzin hydrolase and the sodium-glucose cotransporter 1 (SGLT-1), which transports galactose and glucose.

       Tissue Collection and Blood Chemistry

      When the clinical symptoms of NEC were clearly observed in 1 or more pigs, the entire litter was killed within 8–10 hours (sodium pentobarbitone, 60 mg/kg, IV). This approach allowed us to compare piglets that were clinically healthy (but at risk of developing clinical symptoms of NEC) with those that were clearly pathologic. Before tissue collection, an arterial blood sample was taken and analyzed immediately for pH, partial carbon dioxide pressure, O2, O2 saturation, glucose, hematocrit, hemoglobin, and electrolytes using an automated blood gas and electrolyte analyzer (NOVA Biomedical, Waltham, MA) and a hemoximeter (Radiometer, Copenhagen, Denmark). Blood samples taken from pigs with terminal NEC symptoms were excluded. Plasma was separated and stored (−20°C) for later measurements of cortisol by ELISA (Biomar Diagnostics, Marburg, Germany) and for radioimmunoassays to quantify plasma haptoglobin
      • Petersen H.H.
      • Nielsen J.P.
      • Jensen A.L.
      • Heegaard P.M.
      Evaluation of an enzyme-linked immunosorbent assay for determination of porcine haptoglobin.
      and GLP-2.
      • Petersen Y.M.
      • Hartmann B.
      • Holst J.J.
      • Le Huerou-Luron I.
      • Bjornvad C.R.
      • Sangild P.T.
      Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development.
      Following death, the entire GIT was rapidly removed, and the small intestine, from the pyloric sphincter to the ileo-colonic junction, was isolated by cutting along the mesenteric border and weighed without contents. Intestinal length was measured in a relaxed state on a tabletop and divided into 3 segments of equal length, which were designated proximal, middle, and distal small intestine. The lungs, liver, spleen, heart, adrenals, kidneys, stomach, and pancreas were removed, and the wet masses were recorded. Tissue samples from the pancreas, the stomach (fundus region), the 3 regions of small intestine, and the proximal colon were frozen in liquid nitrogen and stored at −70°C for later biochemical and microbiologic analyses. Three intestinal sections were collected from the middle of each intestinal region. A 10–15-cm section was used for measuring rates of macromolecule and nutrient absorption. Another 10-cm adjacent section was opened along its length for measurements of intestinal circumference, wet mass, and percentage of mucosa that could be removed by gentle scraping with a plastic slide. The proportion of mucosa was determined on a dry matter basis after drying both the mucosa and the underlying tissues (50°C for 72 hours). Additional sections from each region were frozen for biochemical and microbiologic studies. Finally, a 1–2-cm section was fixed in 4% buffered formaldehyde for 2–3 days and then stored in 70% alcohol for later histologic evaluation and for in situ hybridization using specific bacterial probes. For villous height and crypt depth measurements, the samples were embedded in paraffin, sectioned (5 μm), and stained with eosin and hematoxylin.
      • Jensen A.R.
      • Elnif J.
      • Burrin D.G.
      • Sangild P.T.
      Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
      Intestinal morphometry data were collected only from tissue sections that had intact villi without extensive atrophy or necrosis.

       Digestive Enzymes

      The intestine and colon samples were homogenized in 1.0% Triton X-100 (6 mL per g tissue) and the homogenates assayed for disaccharidase (lactase, maltase, sucrase) and peptidase (aminopeptidases N, aminopeptidase A, dipeptidylpeptidase IV) activities using specific substrates, as described previously.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Sangild P.T.
      Glucagon-like peptide 2 enhances maltase-glucoamylase and sucrase-isomaltase gene expression and activity in parenterally-fed premature neonatal pigs.
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      Frozen pancreas was homogenized in Tris-HCl buffer (100 mmol/L containing 20 mmol/L CaCl2, pH 7.9, 2 minutes, 0°C) and used for analysis of amylase, trypsin, and chymotrypsin activity using enzyme-specific substrates.
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      For all measured enzyme activities, a hydrolytic rate of 1 μmol of substrate released per minute at 37°C was equal to 1 unit (U) of enzyme activity. Enzyme activities were expressed per gram of intestine (proximal, middle, distal). Total hydrolytic capacity was estimated by multiplying with the regional intestinal weights and summed for the 3 regions. The concentration of prochymosin in stomach tissue was measured as an indicator of stomach protease synthetic capacity. Fundic tissue was extracted, and prochymosin levels were determined using immunoelectrophoresis and monospecific antisera.
      • Sangild P.T.
      • Schmidt M.
      • Elnif J.
      • Bjornvad C.R.
      • Buddington R.K.
      Prenatal development of the gastrointestinal tract in pigs and the effect of fetal gut obstruction.
      Finally, the tissue messenger RNA (mRNA) level of one intestinal enzyme, maltase-glucoamylase, was measured by quantitative real-time polymerase chain reaction (PCR) and expressed relative to β-actin mRNA levels, as described previously.
      • Bjornvad C.R.
      • Schmidt M.
      • Petersen Y.M.
      • Jensen S.K.
      • Offenberg H.
      • Elnif J.
      • Sangild P.T.
      Preterm birth makes the immature intestine sensitive to feeding-induced intestinal atrophy.

       Intestinal Macromolecule and Nutrient Uptake and Electrochemical Tissue Resistance

      The segments of proximal, middle, and distal small intestine were everted immediately after collection, and 1-cm segments were secured onto stainless steel rods that approximated the intestinal diameter.
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      • Sangild P.T.
      • Schmidt M.
      • Elnif J.
      • Bjornvad C.R.
      • Buddington R.K.
      Prenatal development of the gastrointestinal tract in pigs and the effect of fetal gut obstruction.
      Only tissues that were not necrotic were used for measuring macromolecule and nutrient absorption. Some of the everted sleeves from each of the 3 segments of small intestine were incubated for 5 minutes in 37°C porcine colostrum whey containing bovine IgG or BSA as macromolecule markers (5 g/L). Accumulation of macromolecule by the mucosa was determined by immunoelectrophoresis, as described previously.
      • Sangild P.T.
      • Schmidt M.
      • Elnif J.
      • Bjornvad C.R.
      • Buddington R.K.
      Prenatal development of the gastrointestinal tract in pigs and the effect of fetal gut obstruction.
      Additional sleeves from each region were incubated for 2 minutes in 37°C solutions containing 50 mmol L-1 nutrient concentrations to measure rates of uptake.
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      • Sangild P.T.
      • Schmidt M.
      • Elnif J.
      • Bjornvad C.R.
      • Buddington R.K.
      Prenatal development of the gastrointestinal tract in pigs and the effect of fetal gut obstruction.
      The apical absorption of nutrients was quantified by adding trace amounts of 14C-D glucose with 3H-L glucose or 3H-labeled amino acids or 3H-labeled dipeptide (glycylsarcosine; Gly-Sar) with 14C polyethylene glycol (molecular weight 4000). The amino acids studied (leucine, lysine, proline) are substrates for 3 different carrier systems (neutral, basic, and imino). The peptide bond of Gly-Sar is resistant to hydrolysis by the apical membrane peptidases, and the peptide is absorbed intact by the peptide transporter PEPT1. Rates of glucose absorption represent active transport mediated by the apical membrane sodium-dependent carrier SGLT-1. The mRNA level of this transport protein was also determined by real-time PCR, as described previously.
      • Bjornvad C.R.
      • Schmidt M.
      • Petersen Y.M.
      • Jensen S.K.
      • Offenberg H.
      • Elnif J.
      • Sangild P.T.
      Preterm birth makes the immature intestine sensitive to feeding-induced intestinal atrophy.
      Absorption rates for the amino acids and Gly-Sar include carrier-mediated and carrier-independent pathways. Rates of absorption were normalized to wet tissue weight (nmol · mg-1 · min-1), and total absorptive capacity was estimated by multiplying rates of absorption in each region with regional intestinal weight normalized to body weight.
      Electrochemical transport characteristics were measured essentially as described previously, using intestinal tissues that were stripped of their muscularis layers and mounted in Ussing chambers.
      • Sangild P.T.
      • Holtug K.
      • Diernæs L.
      • Schmidt M.
      • Skadhauge E.
      Birth and prematurity influence intestinal function in the newborn pig.
      The short-circuit current (reflecting the net ion transport) and the tissue resistance (reflecting tissue integrity and permeability) were recorded both under basal conditions and after addition of either phloridzin (1 mol/L), an inhibitor of SGLT-1, or theophylline (5 mmol/L), a secretagogue reflecting the tissue sensitivity to enterotoxin. Finally, Ussing chamber experiments on intestinal tissues in vitro were also applied to test the tissue permeability to the macromolecules ovalbumin and FITC-dextran 4400, using mannitol as the surface marker following methods described elsewhere.
      • Radberg K.
      • Biernat M.
      • Linderoth A.
      • Zabielski R.
      • Pierzynowski S.G.
      • Westrom B.R.
      Enteral exposure to crude red kidney bean lectin induces maturation of the gut in suckling pigs.

       Intestinal NOS Activity and α-Tocopherol Levels

      Intestinal tissue samples were used for determining activities of constitutive NOS (cNOS) and inducible NOS (iNOS) by measuring the conversion of L-14C-arginine into L-14C -citruline, as described.
      • Guan X.
      • Stoll B.
      • Lu X.
      • Tappenden K.A.
      • Holst J.J.
      • Hartmann B.
      • Burrin D.G.
      GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets.
      To determine total NOS (tNOS) activity (nmol per min per g tissue), 2 mmol/L CaCl2 replaced the 4 mmol/L EGTA used in the reaction mixture for iNOS activity. cNOS activity was calculated by subtracting iNOS activity from the tNOS activity. Endothelial NOS (eNOS) mRNA level was measured quantitatively by real-time PCR analysis.
      • Bjornvad C.R.
      • Schmidt M.
      • Petersen Y.M.
      • Jensen S.K.
      • Offenberg H.
      • Elnif J.
      • Sangild P.T.
      Preterm birth makes the immature intestine sensitive to feeding-induced intestinal atrophy.
      The antioxidant α-tocopherol (E vitamin) was determined in both plasma and intestinal tissue extracts (middle region) by high-performance liquid chromatography (HPLC) using saponification with potassium hydroxide (KOH) and extraction into heptane.
      • Knarreborg A.
      • Lauridsen C.
      • Engberg R.M.
      • Jensen S.K.
      Dietary antibiotic growth promoters enhance the bioavailability of α-tocopheryl acetate in broilers by altering lipid absorption.
      Results were expressed as μg α-tocopherol per milliliter of plasma or per gram of intestinal tissue.

       Microbiology

      Conventional culture-dependent microbiology was performed on colonic contents from pigs in experiment 1. Semiquantitative estimation of the number of coliforms was done using MacConkey agar and aerobic culture conditions whereas the number of Clostridia was enumerated using blood agar and anaerobic conditions. The densities of coliforms (lactose-positive colonies on MacConkey agar) and CP-like colonies (colonies with double hemolysis on anaerobic blood agar) were expressed per milliliter contents.
      Treatment differences in the mucosal microbial diversity were elucidated by T-RFLP analysis of DNA extracts prepared from the intestinal mucosa of pigs in experiments 1 and 3 (COLOSTRUM: n = 16; FORMULA: n = 22). We used mucosa of the distal intestine because this region was where NEC-like lesions were most frequently found, and bacterial density was relatively high. The DNA extraction and the details of the T-RFLP analysis have been explained in detail elsewhere.
      • Leser T.D.
      • Amenuvor J.Z.
      • Jensen T.K.
      • Lindecrona R.H.
      • Boye M.
      • Møller K.
      Culture-independent analysis of gut bacteria the pig gastrointestinal tract microbiota revisited.
      The DNA was adjusted spectrophotometrically to a concentration of 5 μg DNA mL-1. Four replicate 50-μL PCR mixtures were made from each sample using the primers S-D-Bact-0008-a-S-20 (5′-AGAGTTTGATCMTGGCTCAG-3′) and S-D-Bact-0926-a-A-20 (5′-CCGTCAATTCCTTTRAGTTT-3′). Primer S-D-Bact-0926-a-A-20 was 5′FAM (carboxy-fluorescein-N-hydroxysuccinimide ester-dimethyl sulfoxide) labelled. Two hundred nanograms of purified PCR products were digested overnight at 37°C with 20 U of CfoI (Boehringer, Mannheim, Germany) in 20 μL reaction mixtures. The fluorescently labeled terminal restriction fragments (T-RFs) were analyzed by electrophoresis on an automatic sequence analyzer (ABI PRISM 373 DNA Sequencer; PE Biosystems, Foster City, CA). The T-RFLP profiles were analyzed by BioNumerics (Applied Maths, Kortrijk, Belgium).
      The location and identity of bacteria adhering to or present in the mucosa was investigated using specific bacterial oligonucleotide probes and fluorescent in situ hybridization on formaldehyde-fixed sections of distal small intestine.
      • Jensen T.K.
      • Boye M.
      • Ahrens P.
      • Korsager B.
      • Teglbjaerg P.S.
      • Lindboe C.F.
      • Moller K.
      Diagnostic examination of human intestinal spirochetosis by fluorescent in situ hybridisation for Brachyspira aalborgi, Brachyspira pilosicoli, and other species of the genus Brachyspira (Serpulina).
      Tissues for analyses were obtained from pigs in experiment 3 containing both healthy and sick pigs (COLOSTRUM, n = 10; FORMULA, n = 11; FORMULA/ANTI, n = 6). Tissue sections (3 μm) were deparaffinated in xylene, transferred to ethanol, and circumscribed with a hydrophobic PAP-pen (Daido Sangyo, Tokyo, Japan) prior to hybridization at a temperature of 49°C. For hybridization, we used a general probe targeting all domain bacteria (EUB338, systematic name: S-D-bact-0338-a-A-18, sequence: GCTGCCTCCCGTAGGAGT)
      • Alm E.W.
      • Oerther D.B.
      • Larsen N.
      • Stahl D.A.
      • Raskin L.
      The oligonucleotide probe database.
      and 2 specific probes targeting CP (systematic name: S-S-Cl.perf.-185-a-A-18, sequence: TGGTTGAATGATGATGCC) and Clostridium difficile (systematic name: S-S-Cl.diff.-193-a-A-18, sequence: TGTACTGGCTCACCTTTG). The probes (MWG-Biotech, Ebersberg, Germany) were tested with pure cultures of known bacteria and found to be specific for the target species. The probes were 5′ labeled with either fluorescein isothiocyanate (FITC) (green) or isothiocyanate derivative Cy3 (red). For scanning in situ hybridization of an entire intestinal cross section, the slides were scanned using the ArrayWoRx microarray scanner (Applied Precision, Issaquah, WA). Because the resolution of the scanner was 5.06 microns, the scans had insufficient resolution to distinguish individual bacteria and mainly visualized bacterial microcolonies.

       Data Analyses and Calculations

      The effects of treatment (COLOSTRUM, FORMULA, FORMULA/GNOTO, FORMULA/EGF, FORMULA/GLP-2, FORMULA/ANTI) were evaluated by analysis of variance using the LSD test to detect treatment differences between 2 individual means (SAS/STAT 8.1; SAS Institute, Cary, NC). Litter and pig were included as random variables. Statistical differences between groups in their NEC frequency were tested by the χ2 test. All organ weight data were expressed relative to body weight. Unless otherwise stated, the intestinal data represent mean values for the proximal, middle, and distal small intestine. Values in Figures and Tables are given as means ± SEM or least square means ± SEM. P = .05 was used as the critical level of significance for all statistical evaluations.

      Results

       Experiment 1: Structural and Functional Indices of NEC Development

       Clinical observations and blood chemistry values

      Despite the transfer to heated, oxygenated incubators within 5 minutes of delivery, all of the preterm pigs suffered from a degree of hypothermia postnatally (mean rectal temperature at 6 hours: 36.0°C ± 0.2°C) with normal rectal temperatures reached at around 12 hours after birth (38.3°C–38.8°C). The degree of hypothermia was not correlated with later NEC development. Similarly, arterial oxygenation saturation did not reach 99%–100% until approximately 12 hours from the initial low values recorded immediately after birth (29.7% ± 0.9%, across all pigs). The FORMULA pigs consistently took longer to be ambulatory (18–24 hours) than COLOSTRUM pigs (12–15 hours).
      Blood samples taken at 20–30 hours, prior to the observation of clinical NEC symptoms, revealed no significant differences in standard blood chemistry values between COLOSTRUM and FORMULA pigs, except that blood pH and potassium values were decreased in the formula group, whereas calcium and cortisol levels were increased (Table 2). The circulating level of the liver acute phase protein haptoglobin was reduced in FORMULA vs COLOSTRUM pigs (Table 2). Plasma GLP-2 levels did not differ between FORMULA and COLOSTRUM pigs, but the concentrations in both groups showed a 10-fold increase relative to values in newborn unfed pigs. Finally, the circulating level of the antioxidant α-tocopherol was significantly reduced in the FORMULA pigs, indicating lowered systemic capacity to resist oxidative stress. Within the FORMULA pigs, diagnosis of NEC was not significantly correlated with any of the above blood chemistry values.
      Table 2Blood Chemistry Values for Newborn and 20- to 30-Hour-Old Premature Pigs Fed COLOSTRUM or FORMULA
      NewbornCOLOSTRUMFORMULA
      pH7.38 ± 0.02
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      7.43 ± 0.02
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      7.31 ± 0.03b
      pCO2 (mm Hg)61 ± 3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      52 ± 2
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      57 ± 3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      Glucose (mmol/L)2.8 ± 0.32.6 ± 0.53.3 ± 0.5
      HCt (%)27.4 ± 1.425.8 ± 1.624.4 ± 1.3
      Hb (mg/100 mL)9.1 ± 0.4
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      8.2 ± 0.5
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      7.8 ± 0.4
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      Na (mmol/L)137 ± 1
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      146 ± 3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      148 ± 2
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      Cl (mmol/L)95 ± 1
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      104 ± 3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      105 ± 2
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      K (mmol/L)4.1 ± 0.3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      5.8 ± 0.3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      4.7 ± 0.3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      Ca (mmol/L)1.16 ± 0.20
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      1.10 ± 0.07
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      1.28 ± 0.04
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      Cortisol (ng/mL)66 ± 5
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      99 ± 16
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      210 ± 25
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      Haptoglobin (mg/mL)2.6 ± 0.3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      0.6 ± 0.1
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      GLP-2 (ng/mL)7 ± 2
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      68 ± 9
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      80 ± 8
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      α-tocopherol (μg/mL)0.28 ± 0.03
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      2.65 ± 0.40
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      1.26 ± 0.49
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      NOTE. Values are mean ± standard error of mean. All blood samples were taken before severe NEC symptoms.
      a,b,c Mean values not sharing a common superscript letter are significantly different (P < .05).

       NEC development

      Some of the manifestations of NEC in preterm FORMULA pigs are evident from the photos presented in Figure 1A–F. The earliest observation of NEC was 20 hours after birth and was evident as abdominal pain and distention, lethargy, apnea, and runny, yellowish stool. Abdominal distention (Figure 1A and B) was the most consistent clinical symptom of severe NEC and was present in almost all pigs with NEC that were graded at 3 or above. Unless killed, death would occur within 6–8 hours once abdominal distention was observed. In experiment 1, the onset of NEC occurred 20–30 hours after the start of oral feeding. During this period, 8 of the 14 FORMULA pigs developed NEC (57%), with an average gross pathologic NEC score of 2.4 ± 0.4 (Table 3). None of the COLOSTRUM pigs showed signs of NEC. At the time of tissue harvest, mucosal hemorrhage, edema, and necrosis (Figure 1D and 1E) were most frequently seen in the distal small intestine and colon. However, severe hemorrhage and necrosis were also seen in the stomachs of some pigs (Figure 1F). The stomachs of FORMULA pigs contained liquid (Figure 1F), whereas the stomach contents of COLOSTRUM pigs was a solid curd of clotted milk. The colon often contained FORMULA but never COLOSTRUM.
      Figure thumbnail gr1
      Figure 1Representative photos showing the clinical signs of NEC in preterm pigs: (A) abdominal distention; (B) air-filled intestines shown by radiograph; (C) pneumatosis intestinalis. Hemorrhagic and necrotic lesions were most frequently seen in the distal small intestine and colon (D) and were characterized by extensive edema, hemorrhage, and air bubbles trapped in the intestinal wall (E; healthy section shown at left side). The stomach contents of FORMULA pigs consisted of unclotted fluid (F, left), and some stomachs were edematous, hemorrhagic, or necrotic (F, right). Microscopic evaluation of cross sections of the small intestine from FORMULA NEC pigs showed severe mucosal or transmural necrosis (G), whereas the intestines from healthy FORMULA pigs were mildly atrophic (H), compared with COLOSTRUM pigs, which appeared normal (I).
      Table 3Organ Weight and Intestinal Morphology Data for Preterm Pigs at Time of Tissue Collection 20 to 30 Hours After Birth (means ± SEM)
      NewbornCOLOSTRUMFORMULA
      Body weight (kg)1.04 ± 0.081.05 ± 0.081.14 ± 0.08
      Age (h)<325 ± 223 ± 2
      Daily gain (g/d)−8 ± 5
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      −36 ± 7
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      NEC score1
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      1
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      2.4 ± 0.4
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      Stomach (g/kg)4.13 ± 0.094.25 ± 0.165.06 ± 0.45
      Pancreas (g/kg)0.96 ± 0.051.11 ± 0.061.10 ± 0.05
      Colon (g/kg)5.5 ± 0.4
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      5.8 ± 0.4
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      7.3 ± 0.3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      Small intestine
       Wet weight (g/kg)17.5 ± 1.3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      33.9 ± 0.8
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      31.2 ± 1.8
      Mean values not sharing a common superscript letter are significantly different (P < .05).
       Length (cm)292 ± 10302 ± 17325 ± 23
       Circumference (mm)8.3 ± 0.2
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      11.4 ± 0.5
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      13.6 ± 0.5
      Mean values not sharing a common superscript letter are significantly different (P < .05).
       Dry matter (%)15.4 ± 0.2
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      20.4 ± 0.7
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      16.0 ± 0.3
      Mean values not sharing a common superscript letter are significantly different (P < .05).
       Mucosa proportion (%)67.2 ± 2.1
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      79.6 ± 1.0
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      69.8 ± 1.4
      Mean values not sharing a common superscript letter are significantly different (P < .05).
       Villus height (μm)573 ± 34
      Mean values not sharing a common superscript letter are significantly different (P < .05).
      312 ± 58
      Mean values not sharing a common superscript letter are significantly different (P < .05).
       Crypt depth (μm)60 ± 364 ± 2
      NOTE. Values are means ± standard error of mean.
      a,b,c Mean values not sharing a common superscript letter are significantly different (P < .05).

       Organ weights and intestinal structure

      The pigs lost weight between delivery and time of death, but slightly more for FORMULA vs COLOSTRUM pigs (Table 3). However, the mass of the small and large intestines of both groups of fed pigs increased 80%–120% during the immediate postnatal period (P < .001). Although intestinal length did not differ between treatments, circumference was greater for FORMULA pigs and the proportion of mucosa and dry matter was lower (P < .05). Microscopic evaluation of intestines from FORMULA pigs with NEC revealed extensive bleeding, mucosal atrophy, stunted villi, and, in the most severe cases, complete disintegration of mucosa, submucosa, and muscularis layers (Figure 1G). Even FORMULA pigs that appeared clinically healthy (Figure 1H) exhibited villous atrophy in the proximal and distal small intestine (Table 3), but hemorrhage and necrosis were not evident. In contrast, the intestines of COLOSTRUM pigs had normal finger-like villi filled with accumulated dietary protein (Figure 1I).

       Digestive enzymes

      Brush border hydrolytic capacities increased sharply after birth in COLOSTRUM pigs, except for lactase and dipeptidylpeptidase IV (Figure 2A–F, all P < .05). Comparable increases did not occur in the FORMULA pigs, and enzyme activities at the time of tissue harvest were lower compared with COLOSTRUM pigs, except for sucrase (Figure 2A–F). The treatment differences were most pronounced in the distal region (50%–70% lower activities for FORMULA pigs; P < .05). Interestingly, the lower maltase activity in the distal small intestine of FORMULA pigs was associated with significant increase in tissue mRNA level for maltase-glucoamylase (relative abundance, 1.96 ± 0.25 vs 1.12 ± 0.17, respectively; P < .05, as detected by real-time PCR analysis). Low enzyme activities were measured in colonic tissue (data not shown) with values reduced in FORMULA vs COLOSTRUM pigs (40%–50% lower; P < .05), except for sucrase and lactase. Activities of digestive enzymes secreted by the stomach (chymosin) and pancreas (amylase, trypsin, chymotrypsin) did not differ between treatments (data not shown). Among these enzymes, only amylase levels increased in response to feeding (from 11 ± 1 U/g tissue in newborns to 38 ± 5 in both groups of fed pigs, P < .05).
      Figure thumbnail gr2
      Figure 2The total hydrolytic activity of 6 different intestinal brush border enzymes (A–F) measured across 3 different intestinal regions (proximal, middle, distal) and expressed relative to body weight (U/kg body weight, means ± SEM). Means with different lowercase letters are significantly different. Only nonnecrotic tissues were analyzed. FORMULA pigs showed significantly reduced activity for all enzymes except sucrase. The Figure also shows the in vitro mucosal uptake capacity for 3 different nutrients (G–I) across the small intestine (μmol/min/kg body weight, means ± SEM). Mean values not sharing a common lowercase letter are significantly different (P < .05).

       Nutrient absorption

      The in vivo response to the oral lactose challenge at 20–30 hours after birth revealed that the increment in plasma galactose (area under the curve) for FORMULA pigs (0.38 ± 0.26 mg/mL min; Figure 3A) was lower compared with values for COLOSTRUM pigs (1.69 ± 0.26; P < .01). Tissue-specific rates of carrier-mediated glucose uptake in the distal small intestine of FORMULA pigs (0.58 ± 0.07 nmol mg-1 min-1) were lower compared with COLOSTRUM pigs (1.02 ± 0.09, P < .05). The treatment effects were less pronounced in the proximal and midregions (data not presented). Correspondingly, total glucose uptake capacity of the entire small intestine of FORMULA pigs was only 20% lower compared with COLOSTRUM pigs (Figure 2G, P < .20). Despite lower rates and abilities to absorb glucose, the relative abundance of SGLT-1 mRNA values in the distal intestine was higher in FORMULA compared with COLOSTRUM pigs (1.49 ± 0.22 vs 0.87 ± 0.10, respectively; P < .05).
      Figure thumbnail gr3
      Figure 3(A) Plasma galactose levels (means ± SEM) after an oral load of lactose (1.5 g/kg body weight) at 18–24 hours after birth in pigs without NEC symptoms. The increment in plasma galactose levels (area under the curve) was significantly reduced in the FORMULA group (P < .01). (B) The proportion of BSA or HSA absorbed from the intestine of preterm pigs fed COLOSTRUM or FORMULA containing BSA (20 mg/mL) at the first feeding (0 hours) and HSA (20 mg/mL) at 12 hours after birth (means ± SEM).
      In the distal small intestine, the FORMULA pigs had higher tissue-specific uptake of lysine (2.27 ± 0.14 vs 1.60 ± 0.16 nmol · mg-1 · min-1, respectively; P < .05) and proline (2.72 ± 0.12 vs 2.05 ± 0.21 nmol · mg-1 · min-1, respectively) compared with COLOSTRUM pigs (P < .05). This did not, however, result in FORMULA pigs having higher small intestine uptake capacities than COLOSTRUM pigs for leucine (Figure 2H), lysine and proline (data not presented), and glycylsarcosine (Figure 2I). Because of intestinal growth, the fed groups of pigs had higher intestinal uptake capacities for all 3 amino acids and the peptide (P < .05).

       IgG and macromolecule absorption

      Over the first 12 hours after birth, the circulating levels of IgG increased gradually in the COLOSTRUM preterm pigs and reached 8.3 ± 0.8 mg/mL. The systemic infusion of sow’s serum into pigs fed FORMULA resulted in IgG levels of 2.5 ± 0.2 mg/mL at 12 hours. When BSA and HSA were included into each of the diets at 0 hours (BSA at the first feeding) and at 12 hours (HSA), FORMULA pigs had lower postfeeding circulating levels of both markers, indicating a reduced ability to absorb intact protein macromolecules by endocytosis (Figure 3B). However, when clinically healthy intestinal tissues collected from both FORMULA and COLOSTRUM preterm pigs were incubated in vitro in sow’s COLOSTRUM, containing bovine IgG and BSA, the resultant uptake of macromolecule into the mucosa was not significantly different among the groups of preterm newborn, FORMULA, or COLOSTRUM pigs (bIgG: 229 ± 22 μg/g, BSA: 188 ± 11 μg/g, pooled values). Hence, the FORMULA-induced reduction in absorptive capacity results from decreased transfer of macromolecules into the circulation, not reduced apical uptake capacity.

       NOS activities and α-tocopherol

      Figure 4 (left panel) shows that FORMULA feeding elicited a significant increase in tNOS activity, relative to COLOSTRUM feeding, because of higher iNOS activity without any change in cNOS levels. The increased iNOS activity was accompanied by a corresponding increase in eNOS mRNA level in the distal intestine (relative abundance, 2.08 ± 0.19 vs 1.55 ± 0.15, respectively; P < .05). Simultaneously, the level of a major tissue antioxidant, α-tocopherol (E vitamin), was markedly reduced in the FORMULA group, relative to the COLOSTRUM group (Figure 4, right panel).
      Figure thumbnail gr4
      Figure 4Total, inducible, and constitutive NOS activity in the small intestine from preterm pigs fed COLOSTRUM or FORMULA (nmol/min/g, means ± SEM). FORMULA pigs showed significantly higher levels of total NOS activity derived mainly from an increased inducible NOS activity (*P < .05). The FORMULA pigs showed significantly lowered concentration of vitamin E in intestinal tissue (μg/g, means ± SEM).

       Experiment 2: Gnotobiotic Preterm Pigs

      Bacteria were not detected in any of the FORMULA/GNOTO pigs, whereas the COLOSTRUM and FORMULA groups had normal evidence of bacterial colonization. Severe clinical signs of NEC were seen in 5 of the 12 conventional FORMULA pigs within 20–30 hours of birth, and the presence of NEC was confirmed by histopathology. None of the FORMULA/GNOTO (n = 12) or COLOSTRUM (n = 11) piglets that were killed at the same time had any clinical or histopathologic signs of NEC. Compared with FORMULA pigs, both COLOSTRUM and FORMULA/GNOTO pigs had elevated distal villous height (Figure 5A), more intestinal mucosa (Figure 5B), and higher intestinal peptidase activities (Figure 5C and D, data not shown for DPPIV). In contrast, disaccharidase activities in FORMULA/GNOTO pigs did not differ from those of the conventional FORMULA group, and both were lower than those of COLOSTRUM pigs (Figure 5E and F, data not shown for sucrase).
      Figure thumbnail gr5
      Figure 5Mucosal morphology indices (A and B) and brush border enzyme activities (C–F; across 3 intestinal regions expressed as U/g tissue) in littermate gnotobiotic preterm pigs fed FORMULA and in conventional preterm pigs fed COLOSTRUM or FORMULA (LSmeans ± SEM, n = 11 or 12). Means with different lowercase letters are significantly different (P < .01).

       Experiment 3: Responses to Immunization, EGF, and GLP-2

       NEC evaluation, organ weights, and intestinal morphology

      In the untreated FORMULA pigs, the incidence of NEC was similar to that found in experiment 1 (13 out of 20 pigs, or 65%, had a NEC score higher than 1.5). The mean NEC score was significantly higher than in the COLOSTRUM group (2.3 ± 0.2 vs 1.4 ± 0.2, respectively; P < .05). Two of the 14 COLOSTRUM pigs had signs of NEC in the colon region, although no atrophy or hemorrhage was observed in the small intestine. As in experiment 1, nonnecrotic tissue from FORMULA pigs showed significant villus atrophy with mean villus heights being reduced 25% ± 8% (P < .05), compared with COLOSTRUM pigs.
      The FORMULA/ANTI pigs had a significantly lowered NEC incidence, relative to FORMULA pigs (0/6 vs 13/20, respectively; P < .01), and there were no clinical or histopathologic signs of NEC (NEC score = 1.0). They remained clinically similar to COLOSTRUM pigs of comparable age (44 ± 3 hours). COLOSTRUM pigs had high circulating antibody titer values against the β toxin of CP (27.0 ± 1.8 IU/mL) with the lowest values in the 2 COLOSTRUM pigs with NEC (6.6 ± 3.5 IU/mL). There was no detectable anti-β-toxin activity in any of the FORMULA pigs. The serum of immunized sows had a titer value of 6.5 IU/mL and, when infused into FORMULA/ANTI pigs (15 mL/kg) resulted in plasma levels of 1–2 IU/mL. All peptidase activities were similar to those in COLOSTRUM pigs, but the FORMULA/ANTI pigs showed reduced mucosa proportion (−8%), villous height (−27%), and width (−14%) and deeper crypts (+50%) and lowered maltase and lactase activities (−65%) relative to the COLOSTRUM group (all P < .05). To confirm the apparently healthy state of the intestine in FORMULA/ANTI pigs, intestinal tissues from the 3 regions were mounted in Ussing chambers. The electrochemical and barrier function properties were similar between the FORMULA/ANTI and COLOSTRUM pigs based on identical values for basal short-circuit current (85 ± 13 μA/cm2) or tissue resistance (30 ± 3 Ω cm2) and the phloridzin- or theophylline-stimulated short-circuit current. Likewise, there was no difference between COLOSTRUM and FORMULA/ANTI pigs in the proportion of FITC-dextan 4400 (0.029% ± 0.004%), ovalbumin (0.002% ± 0.001%), or mannitol (0.31% ± 0.02%) transferred from the mucosal to the serosal side after 120 minutes of incubation. The above pooled mean values across the 3 intestinal regions were similar to those reported for suckling pigs.
      • Radberg K.
      • Biernat M.
      • Linderoth A.
      • Zabielski R.
      • Pierzynowski S.G.
      • Westrom B.R.
      Enteral exposure to crude red kidney bean lectin induces maturation of the gut in suckling pigs.
      Systemic administration of EGF or GLP-2 did not reduce the NEC incidence, compared with experiment 1 (mean NEC score, 2.3 ± 0.3). Four out of 9 pigs (44%) in each of the hormone-treated groups of formula-fed pigs had an NEC score higher than 1.5 across the 4 regions with the usual range of clinical and histopathologic changes (see Figure 1). Relative to control FORMULA pigs, treatment with EGF or GLP-2 treatment did not affect any of the organ weights or the morphology indices measured in experiment 1 (Table 3). Across the control FORMULA, FORMULA/EGF, and FORMULA/GLP-2 groups, nonnecrotic intestinal tissue showed significantly less mucosa (−10%); shorter (−28%) and narrower (−17%) villi; deeper crypts (+18%); and reduced intestinal aminopeptidase N, aminopeptidase A, and dipeptidyl peptidase IV activities (−28%) relative to COLOSTRUM (all P < .05). The only difference between the 2 groups of hormone-treated pigs and the control FORMULA pigs was that the FORMULA-induced decline in all 3 peptidase activities (relative to COLOSTRUM) was significantly less in the EGF and GLP-2 pigs, compared with the control FORMULA pigs (−20% vs −38%, respectively; P < .05).

       Microbial diversity analyses

      The microbial diversity in distal small intestine mucosa, based on bacterial 16S rDNA, did not differ between pigs fed COLOSTRUM or FORMULA. However, the low bacterial diversity compared with older pigs precluded calculation of meaningful diversity coefficients. Of the 18 identified terminal restriction fragments detected above background, a band at 232 bp accounted for the majority of the total intensity. According to our swine-specific database, this band represented a group of Clostridium species (C longisporum, C barati, C quinii, C celatum, C perfringens) plus 1 species of Eubacterium (E nitritogenes). The intensity of this band was significantly elevated in pigs clearly diagnosed with NEC (Figure 6). Other identified species included Lactobacillus mucosae (263–267 bp), Lactobacillus reuteri (407 bp), and a group of Streptococcus (581 bp).
      Figure thumbnail gr6
      Figure 6The mean relative fluorescent intensity of specific terminal restriction fragments (T-RFs), as measured by the T-RFLP analyses (mean ± SEM) of intestinal mucosal DNA extracts from COLOSTRUM (n = 16) and FORMULA (n = 22) piglets. The graph shows the intensities from tissue samples grouped according to presence or absence of NEC (NEC score above or below 1.5). The T-RF with the highest density represents various Clostridium species (T-RF 232 bp), and the density of this T-RF was significantly elevated in NEC piglets vs healthy pigs (*P < .05).

       Fluorescent in situ hybridization

      Based on the T-RFLP findings, the fluorescent in situ hybridization aimed to investigate the general bacterial and the CP populations associated with the mucosa of the distal small intestine. The distal intestine of COLOSTRUM pigs (n = 10) contained very few colonies that hybridized with the general bacterial probe and the specific probe for CP. Those that were detected were consistently found in and around the intestinal contents (Figure 7A and B). Only a few colonies, including CP, were found associated with the surface villous epithelium (Figure 7C), and they were never located deep into the mucosa itself. In 7 of the 10 COLOSTRUM pigs investigated, a film of lightly fluorescent material lined the dense villous surface (Figure 7A and B). A staining test with Alcian Blue revealed this film to be neither mucous material (which would normally disappear during formaldehyde fixation) nor porcine IgG (as assessed by immunochemical staining), but probably to be hydrophilic, slowly degradable food constituents (eg, milk oligosaccharides) that were attached to the surface of the villous epithelium. In these specimens, luminal bacterial colonies could be observed associated with hydrophilic droplets within the lumen (Figure 7B).
      Figure thumbnail gr7
      Figure 7Representative pictures of the in situ hybridization carried out on formaldehyde-fixed sections from the distal intestine of clinically healthy pigs fed COLOSTRUM (A–C), NEC pigs fed FORMULA (D–F), and healthy FORMULA/ANTI pigs (G–I). Hybridization with a general bacterial probe (red) showed that only a few microcolonies of bacteria were attached to tissues from CLOSTRUM pigs (red arrows, general bacterial probe). A (milk-derived) film was detected along the dense villous surface (A and B). Few scattered colonies of Clostridium perfringens (green arrows) were present in the lumen or along the villous surface in 2 out of 10 tested COLOSTRUM pigs (C). All FORMULA pigs diagnosed with NEC showed intense bacterial overgrowth (red arrows, general bacterial probe). Bacteria were present around a markedly atrophic mucosa (D; bacterial translocation) or deep into a severely necrotic mucosa (E). In some NEC pigs, colonization was dominated by Clostridium perfringens (F; green arrows). Bacterial colonization and attachment were either absent or very limited in the FORMULA/ANTI pigs that had received a broad-spectrum antiserum against Clostridium perfringens toxins and E coli antigens (G, H; general bacterial probe, red arrows). When Clostridium perfringens was detected, the colonies never penetrated the epithelial surface (I; green arrows).
      The mucosa from the investigated FORMULA pigs with clinical symptoms of NEC (n = 6) were all heavily colonized with bacteria. Colonies were consistently located around and within the villous epithelium of tissues that were atrophic (Figure 7D) to severely necrotic (Figure 7E). Bacterial colonies were sometimes seen attached to the serosal surface, indicating intestinal perforation or translocation (Figure 7D). Dense populations of CP were present within or closely associated with the mucosal tissue of 3 of the 6 investigated FORMULA pigs with NEC (Figure 7F). They could not be detected in 3 other FORMULA pigs with NEC.
      Tissues from healthy FORMULA pigs (n = 5) and all FORMULA/ANTI pigs (n = 6) consistently had very few bacterial colonies associated with the mucosal surface, and the few detected were located at the surface of the epithelium (Figure 7G–I, 3 FORMULA/ANTI pigs shown). The mucosa appeared mildly atrophic and more open than for COLOSTRUM pigs (Figure 7A and B). CP species were detected among the colonies in some of the healthy FORMULA pigs, but the bacteria never penetrated the villous surface, and the colonies remained outside the vacuolated immature enterocytes (Figure 7I).

      Discussion

      The maturational changes that occur in intestinal structure and function during the final trimester of gestation are critical for the absorption of oral nutrients provided immediately after birth. Consequently, preterm neonates may show immature nutrient (eg, lactose) digestion and absorption and may be partly intolerant to enteral feeding and susceptible to developing NEC.
      • Shulman R.J.
      • Schandler R.J.
      • Lau C.
      • Heitkemper M.
      • Ou C.N.
      • Smith E.O.
      Early feeding, feeding tolerance and lactase activity in preterm infants.
      • Berseth C.L.
      • Bisquera J.A.
      • Paje V.U.
      Prolonging small feeding volumes early in life decreases the incidence of necrotizing enterocolitis in very low birth weight infants.
      • Caicedo R.A.
      • Schanler R.J.
      • Li N.
      • Neu J.
      The developing intestinal ecosystem implications for the neonate.
      Luminal nutrients are critical to maintain intestinal structure and function, even in preterms.
      • Berseth C.L.
      • Bisquera J.A.
      • Paje V.U.
      Prolonging small feeding volumes early in life decreases the incidence of necrotizing enterocolitis in very low birth weight infants.
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      However, knowledge about the response to enteral food in preterms, and its role in NEC development, has been limited by the lack of an animal model in which NEC develops spontaneously without inducing acute intestinal insults. In this study, we show that a large proportion of preterm pigs, reared in infant incubators and fed a human infant formula, spontaneously develops fatal NEC-like symptoms. The observed structural and functional deficits in the small intestine of preterm pigs fed formula were colonization dependent and may predispose to NEC by inducing nutrient malabsorption and excessive fermentation. In agreement with studies in infants, inflammation and NEC could not be related to a specific pathogen, but CP overgrowth was involved in a large proportion of individual pigs with NEC. Our results suggest that provision of immunologic protection against specific bacterial pathogens, via oral colostrum ingestion or systemic immunization, may have the potential to reduce NEC incidence. Finally, our studies show that short-term treatment with the gut growth factor peptides EGF and GLP-2 does not protect the preterm, FORMULA pig from NEC. Hence, milk-derived EGF or milk-induced GLP-2 release in neonates is unlikely to a play major role in the mucosal protection against NEC, despite the reported efficacy of pharmacologic doses in reducing mucosal atrophy.
      • Dvorak B.
      Epidermal growth factor and necrotizing enterocolitis.
      • Petersen Y.M.
      • Hartmann B.
      • Holst J.J.
      • Le Huerou-Luron I.
      • Bjornvad C.R.
      • Sangild P.T.
      Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development.
      Previous animal models of NEC using pigs or rodents have included some of the key features of the human condition by artificially introducing hypoxia, hypothermia, intestinal ischemia, pathogen colonization, and luminal installation of synthetic diets.
      • Di Lorenzo M.
      • Krantis A.
      Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.
      • Dvorak B.
      Epidermal growth factor and necrotizing enterocolitis.
      • Crissinger K.D.
      Animal models of necrotizing enterocolitis.
      • Hsueh W.
      • Caplan M.S.
      • Qu X.W.
      • Tan X.D.
      • De Plaen I.G.
      • Gonzalez-Crussi F.
      Neonatal necrotizing enterocolitis clinical considerations and pathogenetic concepts.
      • Ewer A.K.
      • Al-Salti W.
      • Coney A.M.
      • Marshall J.M.
      • Ramani P.
      • Booth I.W.
      The role of platelet activating factor in a neonatal piglet model of necrotizing enterocolitis.
      The novelty of our model is that NEC develops spontaneously in a large proportion of newborn animals without any artificial insult to the small intestine, except preterm delivery by cesarean section and formula feeding. Following a mild, spontaneous hypothermia and hypoxia in the immediate postnatal period, similar to conditions for preterm infants, bolus feeding with formula led to significant mucosal atrophy in all pigs within 2 days, and some developed the typical symptoms of infant NEC: abdominal distention, vomiting, lethargy, apnea, bloody stools, and severe lesions of the small intestine (most frequently the distal region) and colon, including pneumatosis intestinalis, hemorrhage, edema, and necrosis of the mucosal as well as the serosal layers. These clinical and histologic signs are strikingly similar to the hallmark signs reported in human preterm infants, although gastric mucosal necrosis is rare in infants.
      • Travadi J.N.
      • Patole S.K.
      • Simmer K.
      Gastric pneumatosis in neonates revisited.
      Factors in colostrum protect against NEC because only 5% (2/38) of all the COLOSTRUM pigs developed NEC symptoms, whereas 53% (26/46) developed NEC among the CONTROL pigs fed formula across the 3 experiments. We speculate that an even greater proportion of preterm pigs would have developed NEC if we had extended the protocol beyond 2 days. However, our protocol was designed to enable study of tissue from both clinically healthy and diseased animals having similar postnatal age. Mucosal atrophy and NEC-like symptoms have never been observed in our previous studies using term pigs fed the same amount and type of formula.
      • Jensen A.R.
      • Elnif J.
      • Burrin D.G.
      • Sangild P.T.
      Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
      One of the prevailing theories in NEC etiology is that malabsorbed nutrients (eg, lactose and hexoses) that pass into the distal intestine provide fermentable substrates for overgrowth of pathogenic bacteria (eg, Clostridium species).
      • Di Lorenzo M.
      • Krantis A.
      Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.
      • Waligora-Dupriet A.J.
      • Dugay A.
      • Auzeil N.
      • Huerre M.
      • Butel M.J.
      Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model.
      • Lin J.
      Too much short chain fatty acids cause neonatal necrotizing enterocolitis.
      We provide support for this hypothesis using a novel spontaneous NEC model. At 92% gestation, the preterm pig intestine is structurally and functionally immature,
      • Sangild P.T.
      • Petersen Y.M.
      • Schmidt M.
      • Elnif J.
      • Petersen T.K.
      • Buddington R.K.
      • Michaelsen K.F.
      • Greisen G.
      • Burrin D.G.
      Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
      • Sangild P.T.
      • Schmidt M.
      • Elnif J.
      • Bjornvad C.R.
      • Buddington R.K.
      Prenatal development of the gastrointestinal tract in pigs and the effect of fetal gut obstruction.
      • Sangild P.T.
      • Holtug K.
      • Diernæs L.
      • Schmidt M.
      • Skadhauge E.
      Birth and prematurity influence intestinal function in the newborn pig.
      and, at this stage, FORMULA feeding further reduces the digestive capacity by inducing mucosal atrophy, brush border enzyme defects, and lowered nutrient and macromolecule uptake, compared with COLOSTRUM feeding, or with term newborn pigs fed similar diets.
      • Jensen A.R.
      • Elnif J.
      • Burrin D.G.
      • Sangild P.T.
      Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
      Interestingly, these adverse formula effects depend on the bacterial colonization taking place during the first day of life and are evident even in tissues that appear clinically healthy. Hence, FORMULA-induced mucosal dysfunction may predispose pigs to maldigestion and nutrient fermentation, potentially leading to inflammatory responses and necrosis.
      In the present study, decreased lactase activity was not likely to be responsible for maldigestion because the FORMULA did not much contain lactose. Rather, insufficient maltase activity may limit digestibility of the maltodextrose (hydrolysed corn starch) in the FORMULA. Moreover, the lower maltase-glucoamylase activity and apical glucose transport, despite increases in mRNA abundances for maltase-glucoamylase and SGLT-1, suggest that the FORMULA was unable to maintain the activity or stability of the corresponding apical membrane proteins. Conversely, COLOSTRUM may have a posttranslational effect on the maltase-glucoamylase and lactase-phloridzin hydrolase enzymes, independent of enterocyte turnover and villous architecture. Although lowered peptidase activities were evident only in FORMULA groups with inflammation and NEC, maltase and lactase activities were reduced also in groups that showed limited or no mucosal atrophy and NEC (FORMULA/ANTI, FORMULA/GNOTO).
      The protein source in our FORMULA diet was a mixture of low-molecular-weight peptides of both vegetable origin and from cow’s milk whey. This could be considered an ideal protein source adapted to low luminal proteolytic activity. On the other hand, this diet did not result in the normal formation of a solid curd (clotting of intact milk casein) in the stomach of FORMULA pigs. The possible lack of this physical stimulation could lead to an altered stomach emptying pattern and an impaired intestinal motility that contributes to nutrient malabsorption and fermentation. A relatively large proportion of medium-chain triglycerides in infant formulas may also facilitate excessive production of short-chain fatty acids, potentially damaging the mucosa.
      • Lin J.
      Too much short chain fatty acids cause neonatal necrotizing enterocolitis.
      The nature and the cause of the escalating mucosal inflammation remains an enigma in NEC research. The elevated tissue iNOS activity and eNOS mRNA levels in the clinically healthy FORMULA pigs support the hypothesis that a rapid increase in iNOS activity plays a role as an early mediator of the inflammatory response.
      • Di Lorenzo M.
      • Krantis A.
      Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.
      • Nadler E.P.
      • Dickinson E.
      • Knisely A.
      • Zhang X.R.
      • Boyle P.
      • Beer-Stolz D.
      • Watkins S.C.
      • Ford H.R.
      Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis.
      Excessive NO production might exacerbate mucosal inflammatory responses, particularly in preterm neonates having an immature mucosal blood flow regulation
      • Dyess D.L.
      • Peeples G.L.
      • Ardell J.L.
      • Tacchi E.J.
      • Roberts W.S.
      • Ferrara J.J.
      • Powell R.W.
      Indomethacin-induced blood flow distribution in premature and full-term piglets.
      and a lowered antioxidant capacity. The latter may result in part from an insufficient hydrolysis and uptake of formula antioxidants such as α-tocopheryl acetate (synthetic E vitamin). On the other hand, we were unable to demonstrate higher levels of the presumed protective constitutive form of NOS (cNOS, mainly of neuronal origin) in the healthy pigs fed FORMULA or COLOSTRUM, and the density of nNOS-containing neurons was diet independent in other studies on preterm pigs.
      • Oste M.
      • Van Ginneken C.J.
      • Van Haver E.R.
      • Bjornvad C.R.
      • Thymann T.
      • Sangild P.T.
      The intestinal trophic response to enteral food is reduced in parenterally fed preterm pigs and is associated with more nitrergic neurons.
      This implies that reduced constitutive NOS activity is not a key component in the prematurity- and FORMULA-induced NEC development.
      Clinical evidence and studies with gnotobiotic quails or rodents
      • Waligora-Dupriet A.J.
      • Dugay A.
      • Auzeil N.
      • Huerre M.
      • Butel M.J.
      Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model.
      • Chan K.L.
      • Ng S.P.
      • Chan K.W.
      • Wo Y.H.
      • Tam P.K.
      Pathogenesis of neonatal necrotizing enterocolitis a study of the role of intraluminal pressure, age and bacterial concentration.
      suggest that bacterial colonization is required for NEC, but conclusive evidence from a relevant spontaneous mammalian model is lacking. The present results document that the inflammatory responses are indeed colonization dependent and are not induced by dietary antigens alone. The signs of villous atrophy and peptidase dysfunction were absent in gnotobiotic preterm FORMULA pigs, suggesting a tight interplay between the dietary and microbial determinants that first cause a moderate mucosal damage and later lead to more severe inflammation and NEC. In this study, neither NEC nor FORMULA feeding was associated with gram-negative endotoxemia (data not shown), acute-phase responses such as elevated haptoglobin levels, or a major shift in the mucosal microbial diversity. However, the T-RFLP analysis did show that a relatively high number of Clostridium species colonized the distal mucosa of FORMULA pigs with NEC. The high capacity of these bacteria to ferment undigested substrates and produce exotoxins
      • Waligora-Dupriet A.J.
      • Dugay A.
      • Auzeil N.
      • Huerre M.
      • Butel M.J.
      Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model.
      • Duffy L.C.
      • Zielezny M.A.
      • Carrion V.
      • Griffiths E.
      • Dryja D.
      • Hilty M.
      • Rook C.
      • Morin III, F.
      Concordance of bacterial cultures with endotoxin and interleukin-6 in necrotizing enterocolitis.
      • Butel M.J.
      • Roland N.
      • Hibert A.
      • Popot F.
      • Favre A.
      • Tessedre A.C.
      • Bensaada M.
      • Rimbault A.
      • Szylit O.
      Clostridial pathogenicity in experimental necrotizing enterocolitis in gnotobiotic quails and protective role of bifidobacteria.
      could play a major role in the later phases of disease. The α and β toxins produced by CP type C are known to play a key role for outbreak of necrotizing enteritis in newborn pigs, and vaccination against these toxins prevents disease.
      • Springer S.
      • Selbitz H.J.
      The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.
      Regardless, in the present study, there was not an unequivocal association between NEC and CP colonization as indicated by fluorescent in situ hybridization. Further studies on more tissues from FORMULA preterm pigs (n > 100) have confirmed this conclusion. On the other hand, clinical evidence of NEC was consistently related with bacterial overgrowth and microbial invasion into an already damaged epithelium, whereas the epithelium from healthy pigs, both COLOSTRUM fed and FORMULA fed, contained very few attached colonies of bacteria. Specifically in the COLOSTRUM pigs, toxin antibodies and other milk antimicrobial factors may play a key role in mucosal defense mechanisms by providing a physical barrier to bacterial attachment or by providing milk oligosaccharides that act as competitive binding sites for pathogens.
      • Caicedo R.A.
      • Schanler R.J.
      • Li N.
      • Neu J.
      The developing intestinal ecosystem implications for the neonate.
      Considering that a single pathogen group, including CP, is unlikely to play the key role in NEC etiology, we tested the efficacy of a well-known commercial vaccine product that combines protection against CP toxins and E coli antigens. All FORMULA pigs received systemic infusions of maternal serum IgG, but it was not until this serum contained high titer values against the above pathogenic factors that the FORMULA pigs remained healthy as assessed by both clinical indices and a series of functional tests. Such antibodies, in combination with other naturally occurring bioactive factors in sow’s COLOSTRUM, may be responsible for protecting COLOSTRUM pigs from NEC. It is interesting that the provision of immunoglobulins systemically also provided protection against luminal bacteria. Conversely, it is conceivable that oral treatment would also be effective if the antiserum is provided in a carrier solution that secures normal enterocyte function, including the endocytotic potential. In further studies, high-dose oral and systemic treatment with antiserum against CP toxins (β and ϵ) reduced but did not prevent NEC in FORMULA pigs (unpublished observations). This underlines that more work is required to identify the most important pathogens and toxins, and it cannot be excluded that these differ among species and clinical conditions. Nevertheless, it remains possible that administration of highly specific pathogen or toxin antisera may reduce the incidence of NEC, especially in COLOSTRUM-deprived neonates. This would avoid the potential negative effects of treatment with broad-spectrum antibiotics on natural colonization patterns and the risk of antibiotic resistance.
      Using preterm cesarean-delivered piglets, this study has shown that diet and microbial colonization interact to induce the intestinal conditions that predispose to NEC. In addition to the spontaneous aspect of this model, another advantage is that the size of premature pigs enables the investigation of other relevant approaches such as the effects of total parenteral nutrition, clinical care, and surgical intervention. The preterm pig enables great flexibility in feeding regimens to examine how the level and composition of dietary nutrients, in combination with the resident gut microflora, contribute to the development of NEC, both during its early and its later phases. This model will facilitate further studies to identify the key bioactive nutrients, gut growth factors, probiotics, and immunomodulating agents that help to eliminate this serious disease of the immature newborn.
      The authors thank Thomas Leser, Anna Siekierska, Bente Synnetsvedt, Ebba de Neergaard Harrison, Søren Munk Jensen, and Bent Aalbaek for scientific support and skilled technical assistance.

      References

        • Shulman R.J.
        • Schandler R.J.
        • Lau C.
        • Heitkemper M.
        • Ou C.N.
        • Smith E.O.
        Early feeding, feeding tolerance and lactase activity in preterm infants.
        J Pediatr. 1998; 133: 645-649
        • Berseth C.L.
        • Bisquera J.A.
        • Paje V.U.
        Prolonging small feeding volumes early in life decreases the incidence of necrotizing enterocolitis in very low birth weight infants.
        Pediatrics. 2003; 111: 529-534
        • Caicedo R.A.
        • Schanler R.J.
        • Li N.
        • Neu J.
        The developing intestinal ecosystem.
        Pediatr Res. 2005; 58: 625-628
        • Caplan M.S.
        • Jilling T.
        The role of polyunsaturated fatty acid supplementation in intestinal inflammation and neonatal necrotizing enterocolitis.
        Lipids. 2001; 36: 1053-1057
        • Di Lorenzo M.
        • Krantis A.
        Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.
        J Pediatr Surg. 2001; 36: 700-705
        • Walsh M.C.
        • Kliegman R.M.
        Necrotizing enterocolitis.
        Pediatr Clin North Am. 1986; 33: 179-201
        • Bell M.J.
        • Ternberg J.L.
        • Feigin R.D.
        • Keating J.P.
        • Marshall R.
        • Barton L.
        • Brotherton T.
        Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging.
        Ann Surg. 1978; 187: 1-7
        • Travadi J.N.
        • Patole S.K.
        • Simmer K.
        Gastric pneumatosis in neonates.
        Paediatr Child Health. 2003; 39: 560-562
        • Nanthakumar N.N.
        • Fusunyan R.D.
        • Sanderson I.
        • Walker W.A.
        Inflammation in the developing human intestine.
        Proc Natl Acad Sci U S A. 2000; 97: 6043-6048
        • Claud E.C.
        • Walker W.A.
        Hypothesis.
        FASEB J. 2001; 15: 1398-1403
        • De La Cochetiere M.F.
        • Piloquet H.
        • Des Robert C.
        • Darmaun D.
        • Galmiche J.P.
        • Roze J.C.
        Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants.
        Pediatr Res. 2004; 56: 366-370
        • Waligora-Dupriet A.J.
        • Dugay A.
        • Auzeil N.
        • Huerre M.
        • Butel M.J.
        Evidence for clostridial implication in necrotizing enterocolitis through bacterial fermentation in a gnotobiotic quail model.
        Pediatr Res. 2005; 58: 629-635
        • Morecroft J.A.
        • Spitz L.
        • Hamilton P.A.
        • Holmes S.J.
        Plasma interleukin-6 and tumour necrosis factor levels as predictors of disease severity and outcome in necrotizing enterocolitis.
        J Pediatr Surg. 1994; 29: 798-800
        • Edelson M.B.
        • Bagwell C.E.
        • Rozycki H.J.
        Circulating pro- and counterinflammatory cytokine levels and severity in necrotizing enterocolitis.
        Pediatrics. 1999; 103: 766-771
        • Finer N.N.
        • Peters K.L.
        • Hayek Z.
        • Merkel C.L.
        Vitamin E and necrotizing enterocolitis.
        Pediatrics. 1984; 73: 387-393
        • Feng J.
        • El-Assal O.N.
        • Besner G.E.
        Heparin-binding EGF-like growth factor (HB-EGF) and necrotizing enterocolitis.
        Semin Pediatr Surg. 2005; 14: 167-174
        • Dvorak B.
        Epidermal growth factor and necrotizing enterocolitis.
        Clin Perinatol. 2004; 31: 183-192
        • Petersen Y.M.
        • Schmidt M.
        • Elnif J.
        • Sangild P.T.
        Glucagon-like peptide 2 enhances maltase-glucoamylase and sucrase-isomaltase gene expression and activity in parenterally-fed premature neonatal pigs.
        Pediatr Res. 2002; 52: 498-503
        • Guan X.
        • Stoll B.
        • Lu X.
        • Tappenden K.A.
        • Holst J.J.
        • Hartmann B.
        • Burrin D.G.
        GLP-2-mediated up-regulation of intestinal blood flow and glucose uptake is nitric oxide-dependent in TPN-fed piglets.
        Gastroenterology. 2003; 125: 136-147
        • Sangild P.T.
        • Petersen Y.M.
        • Schmidt M.
        • Elnif J.
        • Petersen T.K.
        • Buddington R.K.
        • Michaelsen K.F.
        • Greisen G.
        • Burrin D.G.
        Preterm birth affects the gastrointestinal responses to parenteral and enteral nutrition in the newborn pig.
        J Nutr. 2002; 132: 2673-2681
        • James P.S.
        • Smith M.W.
        • Tivey D.R.
        • Wilson T.J.
        Epidermal growth factor selectively increases maltase and sucrase activities in neonatal piglet intestine.
        J Physiol. 1987; 393: 583-594
        • Petersen Y.M.
        • Hartmann B.
        • Holst J.J.
        • Le Huerou-Luron I.
        • Bjornvad C.R.
        • Sangild P.T.
        Introduction of enteral food increases plasma GLP-2 and decreases GLP-2 receptor mRNA abundance during pig development.
        J Nutr. 2003; 133: 1781-1786
        • Springer S.
        • Selbitz H.J.
        The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.
        FEMS Immunol Med Microbiol. 1999; 24: 333-336
        • Jensen A.R.
        • Elnif J.
        • Burrin D.G.
        • Sangild P.T.
        Development of intestinal immunoglobulin absorption and enzyme activity in neonatal pigs is diet-dependent.
        J Nutr. 2001; 131: 3259-3265
        • Sangild P.T.
        • Xu R.J.
        Colostrum.
        in: Pond W.G. Bell A.W. Encyclopedia of animal science. Marcel Dekker, New York2004: 229-231
        • Petersen H.H.
        • Nielsen J.P.
        • Jensen A.L.
        • Heegaard P.M.
        Evaluation of an enzyme-linked immunosorbent assay for determination of porcine haptoglobin.
        J Vet Med A Physiol Pathol Clin Med. 2001; 48: 513-523
        • Sangild P.T.
        • Schmidt M.
        • Elnif J.
        • Bjornvad C.R.
        • Buddington R.K.
        Prenatal development of the gastrointestinal tract in pigs and the effect of fetal gut obstruction.
        Pediatr Res. 2002; 52: 416-424
        • Bjornvad C.R.
        • Schmidt M.
        • Petersen Y.M.
        • Jensen S.K.
        • Offenberg H.
        • Elnif J.
        • Sangild P.T.
        Preterm birth makes the immature intestine sensitive to feeding-induced intestinal atrophy.
        Am J Physiol. 2005; 289: R1212-R1222
        • Sangild P.T.
        • Holtug K.
        • Diernæs L.
        • Schmidt M.
        • Skadhauge E.
        Birth and prematurity influence intestinal function in the newborn pig.
        Comp Biochem Physiol. 1997; 118A: 359-362
        • Radberg K.
        • Biernat M.
        • Linderoth A.
        • Zabielski R.
        • Pierzynowski S.G.
        • Westrom B.R.
        Enteral exposure to crude red kidney bean lectin induces maturation of the gut in suckling pigs.
        J Anim Sci. 2001; 79: 2669-2678
        • Knarreborg A.
        • Lauridsen C.
        • Engberg R.M.
        • Jensen S.K.
        Dietary antibiotic growth promoters enhance the bioavailability of α-tocopheryl acetate in broilers by altering lipid absorption.
        J Nutr. 2004; 134: 1487-1492
        • Leser T.D.
        • Amenuvor J.Z.
        • Jensen T.K.
        • Lindecrona R.H.
        • Boye M.
        • Møller K.
        Culture-independent analysis of gut bacteria.
        Appl Environ Microbiol. 2002; 68: 673-690
        • Jensen T.K.
        • Boye M.
        • Ahrens P.
        • Korsager B.
        • Teglbjaerg P.S.
        • Lindboe C.F.
        • Moller K.
        Diagnostic examination of human intestinal spirochetosis by fluorescent in situ hybridisation for Brachyspira aalborgi, Brachyspira pilosicoli, and other species of the genus Brachyspira (Serpulina).
        J Clin Microbiol. 2001; 39: 4111-4118
        • Alm E.W.
        • Oerther D.B.
        • Larsen N.
        • Stahl D.A.
        • Raskin L.
        The oligonucleotide probe database.
        Appl Environ Microbiol. 1996; 62: 3557-3559
        • Crissinger K.D.
        Animal models of necrotizing enterocolitis.
        J Pediatr Gastroenterol Nutr. 1995; 20: 17-22
        • Hsueh W.
        • Caplan M.S.
        • Qu X.W.
        • Tan X.D.
        • De Plaen I.G.
        • Gonzalez-Crussi F.
        Neonatal necrotizing enterocolitis.
        Pediatr Dev Pathol. 2003; 6: 6-23
        • Ewer A.K.
        • Al-Salti W.
        • Coney A.M.
        • Marshall J.M.
        • Ramani P.
        • Booth I.W.
        The role of platelet activating factor in a neonatal piglet model of necrotizing enterocolitis.
        Gut. 2004; 53: 207-213
        • Lin J.
        Too much short chain fatty acids cause neonatal necrotizing enterocolitis.
        Med Hypotheses. 2004; 62: 291-293
        • Nadler E.P.
        • Dickinson E.
        • Knisely A.
        • Zhang X.R.
        • Boyle P.
        • Beer-Stolz D.
        • Watkins S.C.
        • Ford H.R.
        Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis.
        J Surg Res. 2000; 92: 71-77
        • Dyess D.L.
        • Peeples G.L.
        • Ardell J.L.
        • Tacchi E.J.
        • Roberts W.S.
        • Ferrara J.J.
        • Powell R.W.
        Indomethacin-induced blood flow distribution in premature and full-term piglets.
        J Pediatr Surg. 1993; 28: 1396-1400
        • Oste M.
        • Van Ginneken C.J.
        • Van Haver E.R.
        • Bjornvad C.R.
        • Thymann T.
        • Sangild P.T.
        The intestinal trophic response to enteral food is reduced in parenterally fed preterm pigs and is associated with more nitrergic neurons.
        J Nutr. 2005; 135: 2563-2657
        • Chan K.L.
        • Ng S.P.
        • Chan K.W.
        • Wo Y.H.
        • Tam P.K.
        Pathogenesis of neonatal necrotizing enterocolitis.
        Pediatr Surg Int. 2003; 19: 573-577
        • Duffy L.C.
        • Zielezny M.A.
        • Carrion V.
        • Griffiths E.
        • Dryja D.
        • Hilty M.
        • Rook C.
        • Morin III, F.
        Concordance of bacterial cultures with endotoxin and interleukin-6 in necrotizing enterocolitis.
        Dig Dis Sci. 1997; 42: 359-365
        • Butel M.J.
        • Roland N.
        • Hibert A.
        • Popot F.
        • Favre A.
        • Tessedre A.C.
        • Bensaada M.
        • Rimbault A.
        • Szylit O.
        Clostridial pathogenicity in experimental necrotizing enterocolitis in gnotobiotic quails and protective role of bifidobacteria.
        J Med Microbiol. 1998; 47: 391-399