Effects of Commensal Bacteria on Intestinal Morphology and Expression of Proinflammatory Cytokines in the Gnotobiotic Pig

Abstract

A germ-free neonatal pig model was established to determine the effects of bacterial colonization by different species on small intestinal morphology and proinflammatory cytokine gene expression. Two experimental groups of 16 pigs were aseptically delivered by cesarian section and allocated into 4 gnotobiotic isolators. Pigs were either maintained germ-free (GF), or were orally inoculated with either a single strain of nonpathogenic Escherichia coli (EC) or Lactobacillus fermentum (LF) or conventionalized with adult porcine feces (CV). After 13 days tissue samples were collected at 5 regions corresponding to 5%, 25%, 50%, 75%, and 95% of the small intestine (SI) length. In Experiment 2, the GF isolator became contaminated with Staphylococcus epidermidis (SE). In general, intestinal responses to bacterial colonization were similar among GF, LF, and SE pigs, and intestinal responses in EC pigs were more similar to CV pigs. Responses to bacterial colonization were most pronounced in the distal SI regions (50%-95%), suggesting that nonmicrobial factors may be more important in the proximal SI. Relative to CV pigs, the distal intestines of GF, LF, and SE pigs were characterized by long villi, shallow crypts, increased relative intestinal mass, and decreased lamina propria cellularity, whereas SI morphology was intermediate in EC pigs. Relative expression of proinflammatory cytokines interleukin-1beta (IL-1beta ) and IL-6 generally increased distally in the SI and was highest in EC and CV pigs. We observed regional variation in SI morphology and proinflammatory cytokine expression, which differed with bacterial species. This study demonstrates that bacterial species differentially affect intestinal morphology and expression of proinflammatory cytokines and suggests that neonatal bacterial colonization patterns may have long-term effects on intestinal health and development.

Postnatal morphological development and production of short-chain fatty acids in the digestive tract of gnotobiotic piglets

“…Reduced crypt depth and increased villus length agree with the previous observations in germ-free pigs (Kenworthy, 1970;Thompson, and Trexler, 1971;Wostmann, 1975;Shurson et al, 1990;Shirkey et al, 2005). In the present study villi were the longest in the jejunum and shortest in the duodenum and ileum, whereas crypt depth was shortest in the jejunum and deepest in the duodenum throughout the observation period (Figures 5 and 6).…”

“…The mechanisms affecting intestinal length are unknown, however, it can be hypothesized that increased small intestine length in conventionalized pigs is a compensatory response to the decreased absorptive capacity associated with decreased surface area (decreased villus length) and/ or to direct competition with the microbiota for dietary nutrients. Shirkey et al (2005) observed that in the proximal region of the small intestine, the relative weights for segments from conventional pigs tended to be higher than those from germ-free and monoassociated pigs. This is consistent with the previous reports indicating that compared with germ-free animals, conventionally reared animals experience intestinal "thickening" associated primarily with increased lamina propria cellularity (Miniats and Valli, 1973) as well as thickening of the submucosa and muscular layers (Shurson et al, 1990;Furuse and Okumura, 1994;Gaskins, 1997).…”

“…In germ-free and monoassociated pigs (Shirkey et al, 2005), the relative small intestine length was reduced compared with conventional pigs. The mechanisms affecting intestinal length are unknown, however, it can be hypothesized that increased small intestine length in conventionalized pigs is a compensatory response to the decreased absorptive capacity associated with decreased surface area (decreased villus length) and/ or to direct competition with the microbiota for dietary nutrients.…”

“…Although bacterial colonization is lower in the proximal regions (ranging from 10 1 to 10 4 CFU/g of contents) compared with the distal regions (ranging from 10 9 to 10 1 l CFU/g of contents) of the small intestine in the conventional pigs (Stombeck and Guilford, 1990), regional colonization variation (species number or diversity) cannot explain the marked regional variation in small intestinal morphology observed here in the germ-free pigs. Shirkey et al (2005) suggested that regional variation in morphology, especially in the proximal small intestine, is not entirely dependent on microbial colonization but is also influenced by such non-microbial factors as bile salts, pancreatic secretions, and compounds of dietary origin which would be expected to be in higher concentration and have more contact with mucosal surface in the duodenum.…”

The Impact of Probiotics on the Gastrointestinal Physiology

“…Segmental differences were also found in comparative studies conducted on germ free and conventional animals. Shirkey et al (2006) have reported that villus height was the longest in jejunum of pigs supplemented with Lactobacillus fermentum. On the other hand Shurson et al (1990) reported that germ-free pigs had longer ileal and duodenal villi, but shorter jejunal villi compared with their conventional counterparts.…”