Changes in Bacterial Community Structure in the Colon of Pigs Fed Different Experimental Diets and after Infection with Brachyspira hyodysenteriae

Abstract

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with Brachyspira hyodysenteriae, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.The gastrointestinal tract of pigs is densely populated with bacteria, and the intestinal microbiota has important influence on animal health and growth performance. The lumenal contents of the colon support between 10 10 and 10 11 culturable bacteria per g (wet weight) (1,6,21). The majority of the culturable bacteria in the pig colon are gram-positive, strict anaerobic streptococci, lactobacilli, eubacteria, clostridia, and peptostreptococci (21,27,28). The gram-negative organisms comprise about 10% of the total culturable bacteria. Most isolates belong to the Bacteroides and Prevotella groups (27). The gastrointestinal tract bacterial community structure is susceptible to changes in the diet of the animal. For example, the bacterial community will adapt to the introduction of high levels of dietary fibers by increased growth of bacteria with cellulolytic and xylanolytic activities (8,21,32). Other dietary treatments that are known to affect the intestinal bacterial community are the addition of organic acids to the feed (25) and prefermentation of commercial dry feeds (26). However, the effects of these treatments on the structure of the bacterial community are unknown. Pathogenic bacteria and bacteria that are part of the indigenous microbiota in the pig intestine may interact. Resistance to colonization by pathogens is well known (4), while synergistic relations with one or more indigenous intestinal bacterial species are a prerequisite for the pathogenicity of Brachyspira hyodysenteriae (34) and Lawsonia intracellularis (16). Both species cause severe intestinal disorders. On the other hand, it has been shown previously that the onset of swine dysentery causes a dramatic disturbance of the intestinal bacterial community (24). All these studies are based on culturing of the bacteria. However, comparisons with direct microscopic counts have shown that only part of the intestinal bacteria can...

Culture-Independent Analysis of Gut Bacteria: the Pig Gastrointestinal Tract Microbiota Revisited

“…DNA was extracted from intestinal luminal contents by a bead-beating method as previously described (21). Briefly, 200 mg of intestinal content was suspended in 600 l of phosphate-buffered saline, vortexed, and centrifuged 2 min at 200 ϫ g. After transfer to a new tube, the sample was centrifuged at 12,000 ϫ g for 5 min and the pellet was resuspended in 570 l of TE (10 mM Tris-HCl-1 mM EDTA [pH 8.0]).…”

Elective cesarean delivery affects gut maturation and delays microbial colonization but does not increase necrotizing enterocolitis in preterm pigs

“…Before DNA purification, the supernatant was treated with proteinase K (Qiagen, Hilden, Germany) at 56°C for 1 h. DNA was then purified using cetyltrimethylammonium bromide (32), and after centrifugation of the supernatant/phenol/ chloroform/isoamyolalchohol mixture, the DNA was extracted using a 6% Chelax solution (10%, Bio-Rad, Munich, Germany). The bacterial DNA was stored at Ϫ20°C until used for PCR analysis, as previously described (27), with minor modifications. Briefly, the DNA was amplified with the fluorescently labeled 5ЈFAM (carboxy-fluorescein-N-hydroxysuccinimide ester-dimethyl sulfoxide) forward primer S-D-Bact-0008-a-S-20 (5Ј-AGAGTTTGATCMTGGCTCAG-3Ј) (27), and the reverse primer S-D-Bact-0926-a-A-20 (5Ј-CCGT-CAATTCCTTTRAGTTT-3Ј) (33) for 30 cycles, digested with 20 U of restriction enzyme CfoI (Boehringer Mannheim, Mannheim, Germany) for 3 h, loaded onto a denaturing polyacrylamide gel for electrophoresis, and analyzed on an automatic sequence analyzer (ABI PRISM 373 DNA sequencer, PE Biosystems, Foster City, CA).…”

“…The bacterial DNA was stored at Ϫ20°C until used for PCR analysis, as previously described (27), with minor modifications. Briefly, the DNA was amplified with the fluorescently labeled 5ЈFAM (carboxy-fluorescein-N-hydroxysuccinimide ester-dimethyl sulfoxide) forward primer S-D-Bact-0008-a-S-20 (5Ј-AGAGTTTGATCMTGGCTCAG-3Ј) (27), and the reverse primer S-D-Bact-0926-a-A-20 (5Ј-CCGT-CAATTCCTTTRAGTTT-3Ј) (33) for 30 cycles, digested with 20 U of restriction enzyme CfoI (Boehringer Mannheim, Mannheim, Germany) for 3 h, loaded onto a denaturing polyacrylamide gel for electrophoresis, and analyzed on an automatic sequence analyzer (ABI PRISM 373 DNA sequencer, PE Biosystems, Foster City, CA). The lengths of terminal-restriction fragments (T-RFs) were analyzed using the Bionumerics software package v. 4.0 (Applied Maths, Austin, TX) with special emphasis on identification of fragments differentially expressed among treatment groups.…”

“…Briefly, 200 mg of distal SI homogenate was added to 2-ml screw-cap tubes containing 350 mg of 100-m zirconia-silica beads (Biospec Products, Bartlesville, OK) and 600 l of PBS, and shaken for 4 min at high speed on a minibead beater (Biospec Products). Subsequent DNA extraction was performed as previously described (27), with minor modifications. Before DNA purification, the supernatant was treated with proteinase K (Qiagen, Hilden, Germany) at 56°C for 1 h. DNA was then purified using cetyltrimethylammonium bromide (32), and after centrifugation of the supernatant/phenol/ chloroform/isoamyolalchohol mixture, the DNA was extracted using a 6% Chelax solution (10%, Bio-Rad, Munich, Germany).…”

Effects of feed additives on the development on the ileal bacterial community of the broiler chicken

“…In addition, many of the antibiotics that prevent subclinical infections also enhance growth rate and feed efficiency (George et al, 1982;Elsasser et al, 1997;CDUFA, 1999); however, the mode of action for growth promotion has not been fully elucidated (Walton, 1982;Leser et al, 2000). In some instances, it has been shown that AGP's inhibit the growth of pathogenic bacteria, which may cause enteritis and a reduction in feed efficiency (George et al, 1982;Hofacre et al, 1998).…”