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ANIMAL NUTRITION |
* Department of Animal Science, and 2Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada; and
Cargill Animal Nutrition, Minneapolis, Minnesota 55440-5614
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-acid glycoprotein, tumor necrosis factor-, intestinal ammonia, pH, or VFA. However, the PC and DFM treatments showed decreased (P < 0.05) scours at 24 h postinfection compared with the NC, SDPP, and SDPP + DFM diets. Mortality in the NC treatment, which did not contain antibiotics, was greater (P < 0.05) than in the other treatments. Terminal restriction fragment length polymorphism analysis of the 16S rDNA genes of digesta showed a greater incidence (P < 0.05) of Bacteroidetes in the PC and DFM diets than in the NC diet. When SDPP and DFM were included in the diet, the incidence of Bacteroidetes was also greater than in the NC diet (P < 0.05).
Key Words: antibiotic • Escherichia coli • microbial ecology • piglet • probiotic • terminal restriction fragment length polymorphism
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Elimination of in-feed antibiotics is a topical area of research because of the perceived risks posed to human health by animal agriculture via the emergence of antibiotic-resistant bacteria (Heuer et al., 2006). There is a continued need to evaluate alternatives to antibiotics and their modes of action to facilitate improvements in nursery pig nutrition.
Although several studies have evaluated numerous substitutes for antibiotics, few have been assessed within the context of an infectious agent in the digestive tract, and likely explain the variability of success with many products (Fairbrother et al., 2005; Girard et al., 2006). Recent studies have demonstrated the suitability of an Escherichia coli K88+ challenge model in evaluating the role of feed additives in nursery pig diets (Owusu-Asiedu et al., 2002, 2003a,b); however, examination of feed supplements using this model within the context of the microbial ecology of the digestive tract are limited. The microbial composition of the gut is highly complex and is colonized by a large majority of microorganisms that have not been cultured (Krause et al., 2006). Given the central role that gut microorganisms play in resistance to disease (Krause et al., 2006), and thus postweaning health, a more complete understanding of the microbial ecology of the gut in response to antibiotics and potential alternatives to antibiotics would facilitate a mechanistic understanding of postweaning diarrhea in weaned pigs.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The experimental protocol was reviewed and approved by the University of Manitoba Animal Care Committee according to the guidelines of the Canadian Council on Animal Care (1993). A total of 135 Cotswold piglets weaned at 17 ± 1 d were obtained from the University of Manitoba’s Glenlea Swine Research Center. The pigs were weighed and assigned to outcome groups based on the litter of origin and BW. A randomized complete block design constituting 6 treatments, 3 pigs per pen, 2 blocks, and 3 replicates per block was used. This gave a total of 108 pigs that were put on trial, because 27 pigs did not meet the inclusion criteria. The experiment was conducted in 2 blocks, approximately 2 mo apart, because a sufficient number of pigs that met our inclusion criteria were not available in 1 block. The animals were housed in a Biohazard Level 2 animal facility that restricted access to unauthorized personal, and all individuals using the facility were trained in procedures related to biohazard containment. Pigs were housed in animal rooms within pens (1.5 x 1.2 m) with a plastic-covered expanded metal floor. Animals had unlimited access to feed and water throughout the 2-wk study period, with the room temperature set at 29 ± 1°C.
Experimental Diets
All experimental diets were corn and soybean meal based and formulated to meet the NRC (1998) nutrient requirements for piglets weighing 7 to 12 kg (Table 1). Diet 1 was a negative control containing no antibiotics (NC). Diet 2 was the positive control and included the same ingredient composition as NC except that antibiotics (ASP-250, Alpharma, Fort Lee, NJ) were added (PC). Diet 3 was equal to the NC, but with a Bacillus subtilis-based, direct-fed microbial (DFM). Diet 4 was the NC to which spray-dried porcine plasma was added (SDPP). Diet 5 was the NC plus a combination of SDPP and DFM (SDPP + DFM). Diet 6 was the NC plus a combination of supplements, including SDPP and a blend of organic acids, DFM, and a sweetener (Blend; Table 1). The diets were mixed 1 wk before the beginning of each block, using the same batch of ingredients.
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An E. coli K88+ strain was obtained from Carlton Gyles, University of Guelph, and was maintained aerobically in Luria Bertani (LB) broth at 39°C. For piglet dosing, a 5-mL preculture of E. coli K88+ was scaled up by inoculating 2 L of LB broth with the preculture and allowing it to incubate for 16 h at 39°C with agitation. After incubation, a subsample from the 2 L of broth was taken and serially diluted (10-fold), and dilutions of 10–6, 10–7, 10–8, 10–9, and 10–10 were plated on LB agar and allowed to incubate aerobically at 39°C for 16 h. The greatest 2 dilutions at which growth was seen were counted to calculate the cell density of the inoculant.
On d 7 of the experiment, pigs (24 d old) received 6 mL (6.3 x 109 cfu/mL) of the freshly grown E. coli inoculant in the back of the oral cavity by using a syringe attached to a polyethylene tube. The bacteria-rich solution was slowly dribbled into the pig’s throat so that the swallowing reflex was triggered and passage of the inoculant into the lungs was minimized. The severity of diarrhea was quantified by using the fecal consistency scoring method of Marquardt et al. (1999). Fecal scoring (0, normal; 1, soft feces; 2, mild diarrhea; and 3, severe diarrhea) was performed in a blinded fashion by 2 trained personnel with no prior knowledge of the dietary treatments. The presence of blood in the feces was checked daily.
Blood Sampling and Blood Parameter Measurements
Blood was sampled on d 0, 7, and 14 of the experiment via jugular venipuncture by using heparinized vacuum container tubes (Becton Dickinson, Rutherford, NJ) and centrifuged at 3,000 x g for 10 min at 4°C to recover the plasma. Plasma samples were stored at –70°C until required for plasma urea nitrogen determination by a Nova Stat profile M blood gas and electrolyte analyzer (Nova Biomedical Corporation, Waltham, MA). An additional blood sample was collected at the time of slaughter (d 14) via cardiac puncture and centrifuged at 3,000 x g for 10 min at 4°C to recover the serum. Commercial test kits (Cardiotech Services Inc., Louisville, KY) were used to measure -acid glycoprotein, interferon-
(INF-), and tumor necrosis factor-.
Chemical Analysis
Dietary DM was determined by the standard AOAC (1994) method. Crude protein was quantified by a Leco NS 2000 Nitrogen Analyzer (Leco Corporation, St. Joseph, MI). Gross energy was measured with a Parr adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, IL).
Tissue and Digesta Sampling and Histological Measurements
At slaughter, a pig closest to the mean BW of the pen was held under general anesthesia and euthanized by intracardiac injection of sodium pentobarbital (50 mg/kg). Segments of the colon and ileum were removed and placed in sterile containers before transportation to the laboratory for microbial analysis. Additional intestinal segments of approximately 10 cm were obtained from the duodenum, jejunum, and ileum and were fixed in Carnoy’s solution for histological measurements according to the procedure of Owusu-Asiedu et al. (2003b).
Digesta samples were taken from the jejunum, ileum, and colon, and the pH (AB 15, Fisher Scientific, Pittsburgh, PA) was measured. Subsamples (–5 g) from each gut segment were mixed with 5 mL of 0.1 M HCl to stop microbial activity and were stored at –25°C until analyzed for VFA and lactic acid by gas chromatography as described by Erwin et al. (1961). Ammonia nitrogen concentration was measured by using the indole-phenol blue method (Novozamsky et al., 1974).
Microbial Measurements
Culture-Based Analysis.
The ileum and colon tissue samples taken for microbial analysis were weighed (
10 g) and washed vigorously with sterile physiological saline to remove nonattached bacteria. A blunt knife was used to scrape the mucosa down to the connective tissue, and the mucosa was then weighed and diluted 10-fold with anaerobic dilution solution and plated as described previously (Krause et al., 1995). In brief, 10-µL droplets of medium were pipetted onto chromogenic E. coli/Coliform Media (Oxoid, Nepean, Ontario, Canada) or de Man, Rogosa, and Sharpe agar (Fisher Scientific, Ottawa, Ontario, Canada). Dilutions from 10–1 to 10–9 were plated, allowed to dry before inversion, and incubated at 39°C for 24 to 48 h.
Extraction of DNA from Digesta.
The DNA was extracted from the ileal contents by using the QIAamp DNA stool Mini Kit (Qiagen, Valencia, CA), with some modifications. The primary modifications were the use of a mechanical disruption to quantitatively lyse the microbial cells and additional purification steps to provide high-quality DNA. In brief, 180 to 220 mg of digesta was lysed by combining 0.4 g of beads (50:50 combination of 0.1-mm glass beads and 1.0-mm Zirconial/silica beads, Biospec Products Inc., Bartlesville, OK) and 500 µL of lysis buffer (500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 4% SDS; Yu and Morrison, 2004) and 500 µL of phenol:choloroform (2:1). The mixture was placed in a bead-beater (Biospec Products Inc.) and allowed to beat for 3 min. The lysed samples were centrifuged at 10,000 x g for 5 min, and the supernatant was removed before mixing it with 160 µL of 5 M NaCl and 110 µL of 10% hexadecyl-trimethylammonium bromide (Sigma-Aldrich, Steinheim, Germany). Samples were placed on ice for 5 to 10 min and then centrifuged for 10 min at 10,000 x g. Supernatants were transferred to new 1.5-mL microfuge tubes with an equal volume of isopropanol added to precipitate the nucleic acid. The tubes were incubated on ice for 30 min and centrifuged for 10 min at 10,000 x g. The resulting nucleic acid-rich pellet was washed with 500 µL of 70% ethanol and subsequently incubated for 15 min at 39°C with 2 µL of RNase to remove RNA, and 15 µL of proteinase K (20 mg/mL) was added to digest residual proteins (Qiagen). After incubation, 200 µL of AL buffer (Qiagen) was added to the microfuge tube, which was vortexed for 15 s and incubated for 10 min at 70°C. The contents were briefly centrifuged before adding 200 µL of ethanol (95%) to the lysate and were subsequently transferred to QIAamp DNA stool Mini Kit filters. The manufacturer’s protocol was followed from this point on.
Terminal Restriction Fragment Length Polymorphism.
Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to assess the changes in microbial composition in the gut, as described by Abdo et al. (2006). Primers 27f (5'-GAAGA GTTTGATCATGGCTCAG-3') and 342r (5'-CTGCTGC CTCCCGTAG-3') were used to amplify a highly variable section of the 16S rDNA gene (Lane, 1991). The forward primer was fluorescently labeled (WellRED D4dye, Sigma-Proligo Co., St. Louis, MO) to allow detection of the PCR fragments by capillary electrophoresis. The conditions of the PCR reactions were as follows: 1 cycle of 94°C for 5 min; then 36 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 5 min. To produce terminal restriction fragments (T-RF), the 27 to 342 amplicons were digested by using HhaI (10 µL of PCR product, 10 units of HhaI, 1x HhaI buffer, and 20 µg of bovine serum, New England Biolabs, Ipswich, MA). The mixture was adjusted to a final volume of 20 µL with MilliQ water, and the DNA was digested at 39°C for 3 h. The precise lengths of T-RF were determined on a CEQ 8800 Genetic analysis system (Beckman Coulter Inc., Brea, CA). Six microliters of fluorescently labeled fragments, 26 µL of sample loading solution, and 0.5 µL of a DNA size standard (600 bp for T-RFLP, Beckman Coulter Inc., Brea, CA) were mixed together before application to the capillaries. An electropherogram with peaks of different sizes was obtained for each sample, and each peak represented an operational taxonomic unit.
Diversity and Richness.
Chao2, an incidence-based coverage estimator, the Michaelis-Menten function, and the Shannon and Simpson diversity indices were calculated by using Estimates 7.5 (Colwell, 2005). An upper abundance limit of 5 was used to determine rare or infrequent species. The order of the samples was randomized 500 times for each run to reduce the effects of sample order. Tukey’s multiple comparison test (SAS Inst. Inc., Cary, NC) was applied to detect significant differences among experimental groups.
Bioinformatic Analysis of T-RFLP Data.
Online microbial community analysis software (MiCA, version 3, Department of Biological Sciences, University of Idaho; http://mica.ibest.uidaho.edu/, last accessed Dec. 10, 2007) was used to build a putative reference database of probable T-RF of the gut. For this purpose, we incorporated 16S rDNA clone libraries of near-complete sequences of gut microorganisms found in humans (Eckburg et al., 2005), swine (Leser et al., 2002), mice (Ley et al., 2005), and ruminants (Nelson et al., 2003; Ozutsumi et al., 2005) into MiCA and called it the HQ database. This greatly facilitated analysis by excluding the T-RF that were unlikely to occur in the gut, because only 8 out of 26 recognized phyla and no candidate phyla of note have been found in the digestive tract (Rappe and Giovannoni, 2003; Eckburg et al., 2005; Ley et al., 2005). Primers 27f and 342r plus HhaI restriction digestion were applied to the HQ database in silico so that a reference library for our study could be constructed and exported into the phylogenetic assignment tool (Kent et al., 2003). Concurrently, using T-RFLP data obtained from CEQ software (fragment sizes and peak areas), we developed various profiles of interest with reference to dietary treatments. These libraries were entered into the hierarchical browser of the ribosomal database project (RDP-II; Cole et al., 2005) and converted to GenBank format. The resulting libraries were then assigned to the library compare tool of RDP-II. The T-RF of the same size were, in many cases, ambiguous in their assignment of taxonomic rank. All T-RF with multiple accession numbers were assigned to taxonomic rank according to phylum, class, order, and family.
Calculations and Other Statistical Analyses
Data were analyzed as a completely randomized design by using MIXED procedures (SAS Inst. Inc.). Pen was considered the experimental unit for all response criteria measured. When a significant F-value (P < 0.05) for treatment means was observed in the analysis of variance, treatments were compared by using Tukey’s test. Statistical significance (P < 0.01) for the phylogenetic lineage done in the molecular analysis was calculated by using the LSD multiple comparison test.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Growth performance was not affected by dietary treatment (Table 2). Scouring was lower (P < 0.05) at 24 h postinfection for the PC and DFM treatments compared with the other treatments. Mortality was also greater (P < 0.05) for the NC pigs than pigs in the other treatments. Treatment did not affect plasma urea nitrogen, -acid glycoprotein, or tumor necrosis factor-, but INF- was increased (P < 0.05) at d 14 postinfection for the Blend treatment (Table 3). There was no effect of diet on digestive tract pH, ammonia concentration, or organic acid concentration (Table 4). Liver weight differed (P < 0.05) between pigs fed the NC (193.1 g) and the Blend (256.3 g) treatments (Table 5). Duodenum villus height was greater (P < 0.05) in pigs fed the SDPP treatment compared with that of pigs fed the NC and DFM treatments (Table 5).
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There were no differences in the mucosal-associated E. coli when treatments were compared (Table 6). The abundance of lactic acid bacteria in the ileum also did not differ among treatments. Lactic acid bacterial concentrations in the colonic mucosa were lower (P < 0.05) in pigs fed the PC when compared with pigs fed the DFM, SDPP, and SDPP + DFM diets.
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). This approach revealed that at the phylum level, the treatments differed (P < 0.01; Table 7). The PC treatment had a greater proportion of Bacteroidetes and had a significantly decreased proportion of Firmicutes as compared with the NC treatment.
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), which could be differentiated taxonomically (Table 7
). The T-RFLP profiles clearly indicated the differences in the microbial profiles, particularly for PC. Statistical analysis of the T-RF indicated that differences (P < 0.01) among treatments were primarily found in the phyla Bacteroidetes and Firmicutes. These profiles revealed that pigs fed the NC diet had a less diverse population and shorter peaks than did pigs fed diets containing antibiotics, SDPP, and probiotics. These findings agree with results based on the species richness and diversity indices, which showed that NC had lower richness and diversity (P < 0.05) compared with the PC and all other treatments (Table 8). The DFM, SDPP, SDPP + DFM, and Blend treatments did not differ in their richness and diversity indices.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Yi et al., 2005) have noted that protein source influences animal performance in the E. coli K88+ challenge model. Bosi et al. (2004) compared fish protein to spray-dried animal plasma (SDAP), with and without antibiotics, and observed no improvements in performance with the SDAP diet. This result was also observed for the fish protein-based diets, but medication had a beneficial effect only in the fish protein diets and not in the SDAP diets (Bosi et al., 2004). Yi et al. (2005) compared a diet with SDAP or a diet supplemented with glutamine in an E. coli challenge model and observed a growth performance response only in their nonchallenged positive control. No medication was used. Even though we did not observe performance improvements, we did see differences in fecal scours at 24 h and mortality. Pigs in the NC treatment scoured more than pigs fed SDPP alone at 6 h postinoculation and more than pigs fed DFM alone at 24 h. By 48 h, no differences were observed, indicating that the effects of the challenge model were relatively short term. The observation that scouring was relatively short term probably influenced growth performance results in that differences at 7 d were not observable because pigs would have recovered.
Interferon- is at the interface of the microbial interactions with macrophages (Fagarasan, 2006; Straub et al., 2006), and INF- was greater in the Blend diet compared with the SDPP + DFM and the PC diets. The decreased INF- concentration in the SDPP + DFM diet may be due to the protective function of SDPP glycoproteins, which potentially inhibit enterotoxigenic E. coli adhesion by competing for intestinal glycoprotein receptors in the intestine (Bosi et al., 2004). Some authors have differently interpreted the decreased immune response in nursery pigs when SDPP is added to the diet. Touchette et al. (2002) suggested that the reduced level of immune markers was the result of suppression of the immune system by SDPP.
The Bacillus probiotic resulted in no beneficial effect on pig growth performance, but did result in a reduction in scouring. Scouring is an indictor of animal welfare because the reduction of infected feces reduces the overall pathogen load, odor, and hygiene in the pen (European Commission, 2001). Few studies have evaluated Bacillus as a probiotic in E. coli K88+ postweaning diarrhea. Kyriakis et al. (1999) showed a performance response associated with the feeding of a Bacillus probiotic in weaned swine, but this did not include evaluation of experimentally infected animals. Alexopoulos et al. (2004) demonstrated performance benefits with B. subtilis and Bacillus licheniformis spores as probiotics for sows and neonatal piglets, but experimental infection of animals with E. coli was not evaluated. However, in vitro studies indicated that Bacillus can produce compounds antimicrobial to pathogenic E. coli, and may have beneficial effects on gut functions similar to mucin degradation and interconversion of bile salts (Guo et al., 2006).
In our study, it was difficult to conclude that the Bacillus probiotic produced antimicrobial substances against E. coli because there was no response by E. coli. There was, however, a response in the lactic acid population, which indicates that the effect of SDPP and DFM may not be on the gram-negative, but on the gram-positive population. Both Bacillus and Lactobacillus are gram-positive bacteria and use similar mechanisms to transport nutrients across the cell wall. If Bacillus transports nutrients more efficiently than Lactobacillus, this may explain the reduction in colonic lactic acid bacteria (Saier et al., 2002). More efficient competition by Bacillus for the attachment sites used by E. coli may help explain the reduced scouring and the decline in INF-.
We found no differences in intestinal pH, plasma urea nitrogen, intestinal ammonia, and VFA or in intestinal villus morphology. These criteria were measured to provide an indication of the effect of feed additives on intestinal health. However, these measures may be considered an index of recent events in the gut and are not necessarily good indicators of past infections, unless they were severe (Stephen, 2001; Nagy and Fekete, 2005). In this experiment, the level of disease induced by the E. coli challenge model occurred within a 24- to 48-h period, and by d 14 postinfection the animals were fully recovered.
When fermented inulin, lactulose, wheat starch, and sugar beet pulp were added to the diets of pigs, there was greater microbial species richness and diversity in the colonic samples (Konstantinov et al., 2004). When rats were fed casein-based diets containing barley flour, oatmeal flour, cellulose, or barley β-glucans, there was an increase in the microbial diversity and richness, although the authors did not specifically identify this observation and only reported microbial population similarity values (Snart et al. 2006). Our data indicate an increase in species richness, and to a lesser extent species diversity, in the PC and supplemented diets, suggesting that increased microbial species diversity may be an important mechanism by which the gut is protected by antibiotics.
Even though there were significant differences in microbial diversity, there were no differences in growth and feed intake, most immune responses, gut fermentation, and gut morphology. Although a significant response was not observed in growth and feed intake over a 7-d period, significant responses in mortality and fecal scoring occurred within the first 48 h after infection. The changes in microbial diversity are likely the result of dietary effects, and the observed changes in microbial diversity would probably also have occurred within the first 48 h. We hypothesize that the changes in diversity caused by the dietary supplements resulted in the ability of the pigs to recover quickly from infection, with the net result being that most parameters of gut health were recovered by d 7.
To date, little attention has been given to the meaning of changes in microbial diversity in the gut. However, a model has been developed in macroecology that draws a relationship between diversity and ecosystem stability; the higher the diversity, the more stable the ecosystem (Finke and Denno, 2004; Crutsinger et al., 2006). Generally, as microbial diversity in a soil increases, the invasiveness and persistence of phytopathogens decrease (Berg et al., 2002; van Elsas et al., 2002; Garbeva et al., 2003).
These same principles may possibly be applied to the gut ecosystem. Early studies of an adult pig gut experimentally infected with Serpulina (Treponema) hyodysenteriae indicated a decrease in species richness; however, it was difficult to obtain an exact value because it was derived from culture-based data (Robinson et al., 1984). Reduced gut microbial diversity was associated with a lack of colostrum and the development of necrotizing enterocolitis in preterm pigs (Sangild et al., 2006). Experimental infection of piglets with 3 serotypes of E. coli postweaning was associated with a reduction of coliform population diversity and the emergence of diarrhea (Melin and Wallgren, 2002).
In our studies, we also observed a significant increase in the Bacteroidetes in diets supplemented with antibiotics, SDPP, or DFM, and a decline in Firmicutes. Infection with S. hyodysenteriae resulted in a decrease in Bacteroides spp. (from 12.2 to 3.1%; Robinson et al., 1984), thus paralleling our studies. Firmicutes include both Lactobacillus and Clostridium spp., but Robinson et al. (1984) observed an increase in Clostridium spp. only in S. hyodysenteriae-infected pigs. The directional changes in Bacteriodetes and Firmicutes are not only restricted to swine; similar changes also take place in inflammatory diseases of the human digestive tract (Lepage et al., 2005; Bibiloni et al., 2006). Collectively, these data indicate that increased microbial richness in the gut may account for greater stability in the digestive tract, which results in an enhanced ability to recover from infectious postweaning diarrhea.
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Footnotes |
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2 Corresponding author: denis_krause{at}umanitoba.ca
Received for publication December 16, 2006. Accepted for publication November 27, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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