J. Anim Sci.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J. Anim Sci. 2007. 85:1640-1650. doi:10.2527/jas.2006-662
© 2007 American Society of Animal Science
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Figure 1
Right arrow All Versions of this Article:
jas.2006-662v1
85/7/1640    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by George, S.
Right arrow Articles by Kaushik, R. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by George, S.
Right arrow Articles by Kaushik, R. S.

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Lectin binding profile of the small intestine of five-week-old pigs in response to the use of chlortetracycline as a growth promotant and under gnotobiotic conditions1,2

S. George*,§, Y. Oh*, S. Lindblom{dagger}, S. Vilain*,3, A. J. M. Rosa{dagger},§, D. H. Francis{ddagger},§, V. S. Brözel*,§ and R. S. Kaushik*,{ddagger},§,4

* Departments of Biology and Microbiology, and {dagger} Animal and Range Sciences, and {ddagger} Veterinary Science, and and § Center for Infectious Disease Research and Vaccinology, South Dakota State University, Brookings 57007


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Antibiotics have traditionally been used for growth promotion in the pork industry; however, their use in animal feed has recently been limited because of human health concerns. The intestinal microbiota plays an important role in mediating many physiological functions such as digestion and animal growth. It was hypothesized that use of antibiotics as growth promotants and subsequent variations in intestinal microbiota induce significant changes in the intestinal glycoconjugate composition, which ultimately affects animal growth and disease susceptibility. The aim of this study was to characterize the lectin binding profiles of the ileum of weanling pigs in response to the absence of intestinal microbiota and to the use of the antibiotic chlortetracycline as growth promotant. Eighteen half-sib piglets obtained by cesarean section were divided into 3 treatment groups (n = 6) and maintained as control, antibiotic-fed, and gnotobiotic piglets until 5 wk of age. The glycoconjugate composition of the ileal tissues was examined by lectin histochemistry. Lycopersicon esculentum lectin, Jacalin, Pisum sativum agglutinin, Lens culinaris agglutinin (LCA), and Sambucus nigra lectin showed significant differences (P < 0.05) in binding intensities on the dome and villous epithelium between the treatment groups. Griffonia simplicifolia lectin I, Glycine maxi agglutinin, and Arachis hypogea agglutinin exhibited differences (P < 0.05) between treatment groups in lectin binding on goblet cells. Triticum vulgaris agglutinin, Pisum sativum agglutinin, and LCA showed significant differences (P < 0.05) in binding intensities on dome, corona, and follicular regions of the ileum among treatment groups of animals. Overall, ileal tissues from gnotobiotic piglets expressed significantly weaker (P < 0.05) lectin binding for many lectins compared with control and antibiotic groups. This suggests that the intestinal microbiota plays an important role in the expression of sugar moieties in the intestine. Lectins LCA, Phaseolus vulgaris Leucoagglutinin, and Maackia amurensis lectin II showed significant differences (P < 0.05) in lectin bindings between control and antibiotic-fed piglets. This indicates that chlortetracycline as a growth promotant induces biologically relevant changes in the lectin binding profile of the ileum. These findings will help in further understanding the role of the gut microbiota and the mechanisms of action of antibiotics as growth promotants in pigs.

Key Words: chlortetracycline • gnotobiotic • growth promotant • intestinal microbiota • lectin • pig


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Antibiotics, when used in animal feeds as growth promotants, improve growth rate and the efficiency of growth, inhibit subclinical infections, and reduce mortality and morbidity (Cromwell, 2002Go). However, the use of antibiotics in animal feed selects for resistant bacterial strains and facilitates the transfer of resistance genes to pathogens (Schwarz and Chaslus-Dancla, 2001Go). Because of these public health concerns, the use of antibiotics as growth promotants in animal feed has been banned in Europe (Brander, 1999Go).

Lectins present in the animal feed may have antinutritional effects (Banwell et al., 1983Go) and may impact metabolism, maturation of the gut, and health of the animals (Pusztai, 1996Go). These biological effects of lectins are mediated through their binding to carbohydrate moieties; therefore, the glycoconjugate composition of the intestine may influence the growth performance of the animals. Preliminary studies in mice and rats suggest that the composition or absence of intestinal microbiota determines the development of some glycoconjugates and mucins in the gut (Freitas et al., 2005Go). As diversity and selectivity of host tissue glycosylation influence the tissue tropism for infections (Dai et al., 2000Go), alterations in the composition of carbohydrates in the gut may influence host growth and susceptibility to enteric pathogens (Mouricout, 1997Go; Freitas et al., 2005Go). We hypothesized that use of antibiotics as growth promotants causes alterations in the composition of the intestinal microbiota that ultimately induce changes in the glycoconjugate composition of the intestine. The lectin-binding patterns in the small intestine of germ-free/gnotobiotic pigs and in response to the use of antibiotics as growth promotants have not been investigated.

Therefore, this study was designed to determine the effect of the intestinal microbiota and the growth promotant antibiotic chlortetracycline on the lectin binding patterns of the ileum of weanling piglets.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Animals and Treatments
The animal experiments and protocols used in this study were approved by the South Dakota State University (SDSU) Institutional Animal Care and Use Committee.

Three healthy sows, tested negative for porcine respiratory and reproductive syndrome virus and rotavirus, were selected from the University Swine Unit at SDSU and inseminated with the semen of the same boar. Half-sib piglets from these sows were obtained 2 to 3 d before the expected parturition date by cesarean section. In total, 18 piglets, 6 from each of the 3 sows, were used for this study, and 2 pigs originating from each sow were assigned at random to gnotobiotic rearing or to nursing by either of 2 foster mothers. Six pigs (2 from each cesarean section-derived litter) were fostered onto each of 2 sows induced to farrow in synchrony with cesarean section delivery of the test animals, and their naturally born piglets were removed. The foster mothers were kept in separate isolation rooms, thus allowing the colonization of intestines of the pigs with the normal intestinal microbiota of the foster mother.

Gnotobiotic pigs were raised in the Department of Veterinary Science gnotobiotic pig facilities, which have been successfully used for various studies (Baker et al., 1997Go; Butler et al., 2002Go, 2005Go). The gnotobiotic piglets were supplied with commercial sterile milk replacer that was prorated based on animal age and expected energy requirements (Esbilac, PetAg Inc., Hempshire, IL) for 21 d. Esbilac milk replacer is composed of water, skimmed milk, soy oil, sodium caseinate, butter, egg yolk, calcium caseinate, L-methionine, L-arginine, calcium carbonate, choline chloride, potassium chloride, lecithin, magnesium sulfate, monopotassium phosphate, salt, tricalcium phosphate, carrageenan, dipotassium phosphate, di-calcium phosphate, ascorbic acid, ferrous sulfate, zinc sulfate, vitamin A supplement, vitamin E supplement, niacinamide, D-calcium pantothenate, copper sulfate, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, manganese sulfate, vitamin D3 supplement, potassium citrate, potassium iodide, folic acid, D-biotin, phytonadione (source of vitamin K), and vitamin B12 supplement.

After 21 d, piglets were weaned or removed from milk replacer and then fostered piglets were regrouped as control (antibiotic-free) and antibiotic-fed groups (n = 6). The fostered control and antibiotic-fed groups were formed by including 3 piglets from each foster sow. Fostering on nurse sows and redivision at weaning was done to nullify the effect of swine genetics on the intestinal microflora composition of the experimental piglets. All the 3 groups of piglets were fed the same weanling diet (Table 1Go) ad libitum for 2 wk, except for the addition of chlortetracycline at 50 ppm in the feed of the antibiotic-fed group, after which the piglets were euthanized at 5 wk of age and the study was concluded. The growth performance of the pigs in response to the use of chlortetracycline in the feed was not measured in this study. Fecal samples from the gnotobiotic pigs were submitted to the SDSU Veterinary Science Diagnostic Laboratory for testing for aerobic and anaerobic microbes at 2 and 5 wk of age. No aerobic or anaerobic bacteria were detected, except for Clostridium perfringens in the samples collected at 5 wk of age, and hence the term gnotobiotic pig was used in this study.


View this table:
[in this window]
[in a new window]

  		Table 1. Composition of experimental diets (as-fed basis) for weaned control, antibiotic-fed, and gnotobiotic groups of pigs during wk 4 and 5 of the experiment
 
Sample Collection
One-third of the animals from each group were killed on consecutive days for technical convenience. Piglets from each group were euthanized using a standard protocol approved by the Institutional Animal Care and Use Committee. After euthanasia, the abdomen was opened immediately, the mesentery was removed, and the ileum bearing a continuous Peyer’s patch was located. A caudal segment of the ileum was excised, opened longitudinally, and the digesta were removed by gentle washing in PBS (pH 7.3). Distal segments of the ileum, 5 to 10 cm long, were obtained from each piglet and immediately stapled at both ends onto a thick, polyethylene transparency sheet to preserve tissue morphology. All samples were immediately immersed in 10% buffered formalin for 48 h, followed by storage in 70% ethanol. Tissues were embedded in paraffin, and sections 5 µm in thickness were cut and laid on lysine-coated microscope slides (Fisher Scientific, Chicago, IL).

Lectin Histochemistry
We first tested and standardized the lectin histochemistry protocol on ileal sections from control pigs using various lectin concentrations. We found that a lectin concentration (10 µg/mL) that was also used by others on pig intestinal tissues (Chae, 1997Go) provided acceptable staining intensities on control pig ileal tissues; therefore, we used all lectins at 10 µg/mL concentrations in this study. The names of all 23 lectins used in this study and their abbreviations and carbohydrate specificities are provided in Table 2Go.


View this table:
[in this window]
[in a new window]

  		Table 2. Lectins used in this study
 
Briefly, tissue sections (5 µm) were deparaffinized using xylene and hydrated using descending gradients of alcohol (100, 95, 70%) to PBS. Each treatment was carried out for 2 min and all washes were done in PBS for 5 min. Nonspecific protein binding was blocked by treating the sections with 1% goat serum in PBS for 20 min. Sections were washed 3 times in PBS and were incubated with 1 of the 23 biotinylated lectins (BK-1000, BK-2000, BK-3000, Vector Laboratories Inc., Burlingame, CA) for 1 h (100 µL/section, 10 µg/mL). The sections were then washed twice in PBS. Sections that were not treated with lectins served as the negative controls. Endogenous peroxidase was quenched by incubating the sections in 0.3% hydrogen peroxide in PBS for 10 min. The tissue sections were again washed twice in PBS and incubated with Avidin-biotinylated peroxidase complex (Vector Laboratories) for 30 min. After washing 3 times in PBS, the sections were treated with 3,3 '-diaminobenzidine (Vector Laboratories) for 7 min and counter-stained with Mayer’s hematoxylin. The sections were dehydrated through ascending grades of alcohol (95, 100, 100%) to xylene and mounted permanently using Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI). To assess the specific binding of lectins to carbohydrate residues on the ileal tissues, lectins were preincubated with a corresponding group of sugars (Vector Laboratories) for competitive inhibition and then applied to the tissues for staining. All of the sugars for lectin blocking were used at the concentrations (2 to 8 µM) suggested by the manufacturer (Vector Laboratories).

Analysis
Instead of evaluating the whole tissue section for lectin binding, the cross-sections of ileum with Peyer’s patches were divided into 8 compartments, namely the dome epithelial surface, the villous epithelial surface, the villous goblet cells, the crypt epithelial cells, the dome, the corona, the follicle, and the interfollicular area (Chae, 1997Go; Jeong et al., 2002Go), for the convenience of making comparisons, and each region of the tissue was evaluated independently for lectin binding.

Three fields (10 x objective) were scanned from each tissue compartment to make sure that equal areas were included for regional comparisons of ileal tissues from the treatment groups. The compartments were scored based on the intensity of brown coloration as visualized with a light microscope under 10 x magnification. The intensity of lectin bindings was scored as: 0 = no staining, and 1 = weak, 2 = moderate, or 3 = heavy staining for each of the 23 lectins on tissues from all 18 pigs. Thus, histochemistry scoring of lectin binding was subjective; however, all of the measurements were made by a single person blinded to treatments. The background staining was minimal or absent, and there was an obvious differential staining pattern for all of the lectins.

In this study, our major research question was to determine whether gnotobiotic- and antibiotic-fed pigs differed from the control pigs in the expression of carbohydrate moieties in the ileum. Although orthogonal contrasts is a useful and powerful technique for analysis of experimental data for mean comparisons, our data did not fulfill the required criteria or conditions of planned orthogonal contrasts to compare 3 treatment groups as control vs. antibiotic, antibiotic vs. gnotobiotic, and control vs. gnotobiotic. As the ranked data obtained from lectin binding staining from all 3 groups was not normally distributed, data were analyzed using nonparametric ANOVA by Wilcoxon test to compare 3 groups of animals as control vs. antibiotic, antibiotic vs. gnotobiotic, and control vs. gnotobiotic. The JMP IN version 5.1 (Statistical Discovery Software, Thompson/Brooks/Cole, SAS Institute Inc., Cary, NC) was used to analyze the data. A P-value of < 0.05 was considered significant, and the results were expressed as means ± SE.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Lectin Binding Profile of Various Epithelial Surfaces in Pig Ileum
Overall, all of the 23 lectins showed binding in varying degrees on various epithelial surfaces in all 3 piglet groups (control, antibiotic, gnotobiotic, Tables 3Go and 4Go). Except for Lycopersicon esculentum lectin (LEL), Jacalin, Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), and Sambucus nigra lectin (SNA), the lectins did not show significant differences in the binding profiles of the dome epithelium among groups (Table 3Go). Ileal tissues from the gnotobiotic pigs showed reduced binding affinity on dome epithelium for PSA, LCA, and SNA compared with the control and antibiotic groups (Figure 1Go, online data supplement). Lectin LEL stained the dome epithelium of control pigs with more intensity than the gnotobiotic pig tissues, whereas Jacalin showed significant differences in binding intensities between the antibiotic and gnotobiotic groups.


View this table:
[in this window]
[in a new window]

  		Table 3. Lectin binding on the dome and villous epithelial surfaces of the ileum in control, antibiotic-fed, and gnotobiotic pigs1
 

View this table:
[in this window]
[in a new window]

  		Table 4. Lectin binding on villous goblet cells and crypt epithelium of the ileum in control, antibiotic-fed, and gnotobiotic pigs1
 

Figure 1
View larger version (133K):
[in this window]
[in a new window]

  		Figure 1. Histochemical staining of ileal tissues of weanling pigs for lectin binding. Lectin binding profile of (A–C) Pisum sativum agglutinin (PSA) and (D–F) Lens culinaris agglutinin (LCA) in (A and D) control, (B and E) antiobiotic-fed, and (C and F) gnotobiotic pig ileal tissues showed more intense staining in control and antibiotic-fed animals compared with gnotobiotic pigs. Note in (G) that no background staining was obtained on the ileal tissue when no lectin was used in the staining procedure. When PSA and Canavalia ensiformis A (Con A) lectins previously incubated with mannose/glucose sugars were used on ileal tissues, no (H) PSA- or (I) Con A-specific staining was observed. This indicates that both of these lectins specifically bind to sugar moieties on the ileal tissues. Size bar = 100 µm.

 
Except RCA120, LEL, Jacalin, PSA, LCA, and SNA, no lectins showed significant differences in their binding intensities on the villous epithelial surface among pigs of the 3 groups (Table 3Go). The lectins Jacalin, PSA, LCA, and SNA showed less binding affinity on the villous epithelial surfaces of gnotobiotic pigs compared with control and antibiotic groups (Figure 1Go, online data supplement). Lectin RCA120 did not show any significant differences in binding affinities between control and antibiotic groups, but less staining was observed in gnotobiotic pigs compared with the antibiotic group of pigs. By contrast, LEL showed significant differences in the binding intensities between control and gnotobiotic groups, but not between antibiotic and gnotobiotic groups. Only LCA showed significant differences in binding affinities between the control and antibiotic groups.

No significant differences in the lectin staining intensities on villous goblet cells were observed among different groups except for Griffonia simplicifolia lectin I, Glycine maxi agglutinin (SBA), and Arachis hypogea agglutinin (PNA; Table 4Go). Lectin SBA showed significant differences between the control and gnotobiotic groups, and between the antibiotic and gnotobiotic groups, but no difference in SBA binding intensities was observed between the control and gnotobiotic group. Griffonia simplicifolia lectin I and PNA showed significant differences in binding intensities between the antibiotic and gnotobiotic groups. No significant differences in lectin staining intensities on crypt epithelium were observed among the 3 groups (Table 4Go).

Lectin Binding in Subepithelial Peyer’s Patch Regions of Pig Ileum
All of the lectins used in this study except Sophora japonica agglutinin (SJA) stained the dome regions of ileum in piglets of all the groups (Table 5Go). Although average intensity of staining varied from 0.17 to 2.4 among different lectins and among the 3 groups of animals, PSA and LCA showed significant differences in lectin binding intensities between the control and gnotobiotic groups, whereas Triticum vulgaris agglutinin (WGA) and LCA showed significant differences between the antibiotic and gnotobiotic groups. Lectin Phaseolus vulgaris Leucoagglutinin (PHA-L) showed significant differences in binding intensities between the control and gnotobiotic groups.


View this table:
[in this window]
[in a new window]

  		Table 5. Lectin binding on the dome and corona regions of the ileum in control, antibiotic-fed, and gnotobiotic pigs1
 
Four lectins, SJA, Vicia villosa agglutinin, succinylated WGA (s-WGA), and Erythrina cristagalli lectin, did not bind to corona regions of ileal tissues of any pigs of the 3 test groups; however, the other 19 lectins bound to corona regions in pigs of the 3 groups with varying intensities (Table 5Go). No significant differences were observed in lectin binding in the corona region among 3 groups of animals except with lectins WGA, PSA, LCA, PHA-L, and Maackia amurensis lectin II (MAL). The PSA, LCA, and MAL showed significant differences in binding between the control and gnotobiotic groups, whereas WGA and PHA-L showed significant differences in binding between the antibiotic and gnotobiotic groups. In both cases, the gnotobiotic pigs showed less staining compared with the control and antibiotic groups. Staining of the corona region with MAL was only different between the control and antibiotic-treated pigs.

Two lectins, SJA and Vicia villosa agglutinin, did not show any binding to follicles; however, the other 21 lectins showed varying degrees of staining among piglets in the 3 groups (Table 6Go). Lectins WGA, PSA, and MAL showed significant differences in follicular staining among the 3 groups. Lectin MAL-stained follicles in control animals more intensively compared with the antibiotic group. Lectin WGA showed significant differences in follicular staining between the antibiotic and gnotobiotic group, whereas LCA and PSA showed greater intensity of staining in follicles in control compared with gnotobiotic animals.


View this table:
[in this window]
[in a new window]

  		Table 6. Lectin binding on the follicles and interfollicular area (IFA) of the ileum in control, antibiotic-fed, and gnotobiotic pigs1
 
Two lectins, GSL-II and s-WGA, did not stain the interfollicular area (IFA) in ileal tissues in pigs from any of the 3 treatment groups, but the other lectins stained the IFA with varying intensities (Table 6Go). No significant differences in lectin binding among treatment groups to the IFA were observed except with SNA, which showed more intense staining in the gnotobiotic group than the antibiotic group.

Overall, lectins PSA and LCA consistently showed significant differences in lectin binding, both on epithelial surfaces and Peyer’s patches among 3 groups of animals; therefore, representative photographs of tissues strained with these 2 lectins have been included as examples of lectin binding (Figure 1Go, online data supplement).

Specificity of Lectin Binding
In order to assess the specificity of lectin binding to pig ileal tissues, various lectins were incubated with blocking sugars before use in lectin binding studies. Incubation of lectins with relevant blocking sugars reduced or completely blocked their binding to ileal tissues, indicating the specificity of lectin binding to particular sugars on ileal tissues. Representative photographs that show inhibition of lectin binding for PSA and Canavalia ensiformis A have been included in Figure 1Go (online data supplement). Furthermore, absence of binding of certain lectins to ileal tissues or to specific sugars on the same tissue also supports the specificity of lectin binding on pig ileal tissues used in this study.


   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
To our knowledge, this is the first comprehensive study that has been conducted for characterizing the glycoconjugate composition of the ileum of weanling pigs in response to use of the antibiotic chlortetracycline as growth promotant and under gnotobiotic conditions. In this study, half-sib piglets obtained by cesarean section were used to minimize the genetic variation among the experimental subjects. Host genetics has been shown to play an important role on the intestinal microbiota composition (Vaahtovuo et al., 2003Go; Ley et al., 2005Go; Stewart et al., 2005Go). Thus, to avoid or minimize the effect of differences in intestinal microbiota of the host, if any, on the intestinal microbiota composition of experimental animals, piglets kept on 2 foster mothers were segregated into control and antibiotic-fed groups in equal proportions.

Feeding of milk replacer (Esbilac) to gnotobiotic pigs in this study cannot be directly compared with sow colostrum and milk which are rich in growth factors and immunoglobulins (Klobasa et al., 1987Go; Atwood and Hartmann, 1992Go) and may influence the intestinal development and health. However, it was crucial to feed gnotobiotic pigs a sterile diet, and therefore, feeding sow milk was not a feasible and practical approach. Feeding Esbilac was the best available alternative because it had been successfully used in other studies to maintain gnotobiotic pigs (Baker et al., 1997Go; Butler et al., 2002Go, 2005Go). Furthermore, many studies indicate that milk replacer feeding is beneficial to weight gains in pigs (Zijlstra et al., 1996Go; Wolter et al., 2002Go) and may be comparable with sow milk for animal growth. The possibility that feeding milk replacer to gnotobiotic pigs before weaning may impact the carbohydrate expression in the ileum cannot be completely excluded, but this effect was minimized to a greater extent by feeding gnotobiotic pigs the same weanling diets as pigs in the other groups during the last 2 wk of the study.

Among many different antibiotics used as growth promoters, chlortetracycline was chosen for this study for 2 specific reasons. First, chlortetracycline is widely used as feed additive in the pig industry for growth promotion (Ozawa, 1955Go; Cromwell, 2002Go). Second, chlortetra-cycline was found to promote a greater growth rate in pigs compared with tylosin and carbadox in an experiment conducted at SDSU before this study was undertaken (unpublished data), and therefore, the growth performance of pigs was not measured directly in this study. Chlortetracycline was incorporated in the feed at the rate of 50 ppm to match the conditions practiced in pork production (Hathaway et al., 1996Go; Gaskins et al., 2002Go). In a parallel study, we detected the presence of different microbial communities in the ileal contents of control- and antibiotic-fed pigs by 16s rRNA gene sequencing. We observed significant differences in the composition of microbial community in the ileum of these 2 groups of pigs. The intestinal microbial community in both groups was largely composed of members of the low gnotobiotic + control bacteria including various Lactobacillus and Clostridium phylotypes. However, Turicibacter sanguinis and C. lituseburense dropped by more than 50% in chlortetracycline treated pigs. The L. johnsoniae numbers decreased, whereas L. amylovorus gained in dominance to 48% of the community when pigs were fed chlortetracycline (unpublished data). These observations confirmed that feed chlortetracycline was biologically active and induced significant changes in the composition of intestinal microbiota.

In this study, tissue samples were collected from the caudal ileum because the ileum has more intestinal microbiota compared with jejunum, and possibly microbes present in the ileum can exert maximum influence on growth promotion and intestinal glycoconjugate composition (Gaskins et al., 2002Go). The possible role of antibiotics as growth promotants is to function as modulators by helping the intestinal microbiota to adapt to a new diet, and therefore we concluded this study 2 wk after weaning. Earlier studies have indicated that use of antibiotics as growth promotants and absence of intestinal microbiota in pigs induce morphological changes in the gut (Gaskins et al., 2002Go; Shirkey et al., 2006Go); however, morphometric comparisons between treatments were not performed here because it was beyond the scope of the current study.

One of the possible approaches for glycoconjugate quantification is to isolate and measure them directly; however, this approach may not be very practical and feasible when glycoconjugate localization and expression in various cellular compartments of the tissue is the major objective as was the case in this study. This approach also requires cumbersome isolation and digestion procedures. Therefore, a semiquantitative histochemical lectin binding approach which had been successfully used by others (Chae and Lee, 1995Go; Chae, 1997Go; Choi et al., 2003Go; Freitas et al., 2005Go) in assessing the carbohydrate composition of intestinal tissues was preferred for this study.

Glycoconjugate masking by endogenous ligands is a matter of concern especially when formalin-fixed intestinal tissues are used for histochemistry. Furthermore, there is a possibility that some lectins present in the feed may mask the carbohydrate moieties present on the intestinal surfaces and may interfere in their in vitro detection because we did not analyze the presence of specific lectins in the feed in this study. We processed the tissue sections for lectin staining using the procedures that efficiently strip the endogenous ligands from epithelial glycoconjugates and have been successfully used by others (Chae and Lee, 1995Go; Chae, 1997Go; Choi et al., 2003Go; Freitas et al., 2005Go). All 23 lectins efficiently bound (binding intensities of 1 to 3) to the intestinal epithelium of control pigs, confirming that glycoconjugates were freely available on epithelial surfaces for lectin binding. Because all 3 groups of piglets were fed the same diet in the last 2 wk of the study, we did not anticipate that animal feed and treatments differentially affected the degree of glycoconjugate masking in various groups of animals and confounded the interpretation.

Many studies have been conducted earlier to analyze the expression of various carbohydrates on the epithelium and Peyer’s patches in the conventional pig small intestine (Jaeger et al., 1989Go; Brown et al., 1991Go; Gelberg et al., 1992Go; Chae and Lee, 1995Go; Chae, 1997Go; Jeong et al., 2002Go). Conventional animals used in this study showed the similar lectin binding profiles for various lectins as observed in those earlier studies. It is clear from the earlier studies that age and diet of the animal affect the lectin binding profile in the intestine (Jaeger et al., 1989Go; Gelberg et al., 1992Go; Pusztai et al., 1993Go; Pusztai, 1996Go).

In this study, 3 lectins (LCA, PHA-L, and MAL) showed differential binding patterns between control and antibiotic groups in various regions of the ileum. Alterations in the composition and structure of carbohydrates on the gut epithelium may influence the growth of the host, bacterial tropism and susceptibility or resistance of the host to enteric pathogens (Mouricout, 1997Go; Freitas et al., 2005Go). Pathogens like Campylobacter, Escherichia coli, Vibrio, and commensals like Lactobacillus display surface lectins on their pili/fimbriae that help in their adherence to the intestinal tract (Freter, 1981Go; Farthing, 1985Go; Sharon, 1987Go; Mouricout, 1997Go). Enteric viruses like rotavirus and protozoans like Cryptosporidia, Entamoeba histolytica, and Isospora suis frequently attach and invade the intestinal epithelial cells by recognizing carbohydrate moieties on the cell surface (Kelly et al., 2000Go; Sohn and Chae, 2000Go; Sousa et al., 2001Go; Stanley and Reed, 2001Go; Dormitzer et al., 2002Go; Choi et al., 2003Go; Tavares et al., 2005Go). The lower binding intensities of LCA and MAL in antibiotic-fed animals compared with control animals suggest the lower expression of glucose/mannose and sialic acid moieties in the gut of 3 animals. Because glucose/man-nose and sialic acid are involved in the adherence of enterotoxigenic E. coli (Grange et al., 1997Go, 1998Go, 1999Go) and rotaviruses (Mendez et al., 1999Go; Arias et al., 2002Go), respectively, these findings may have implications in lower susceptibility of antibiotic-fed pigs to enteric pathogens. The intense binding of LCA and MAL lectins among control piglets may indicate a more diverse microbial population in the intestine than in the antibiotic group of piglets because antimicrobials as growth promoters may suppress or inhibit the growth of certain microbes, making the small intestinal microbial population more homogeneous (Gaskins et al., 2002Go).

It was our intention to keep the gnotobiotic pigs as germ-free pigs during the whole course of the study; however, although these pigs remained germ-free up to 2 wk of age, they were found to be contaminated with low levels of C. perfringens at 5-wk of age. We could not determine whether these pigs got contaminated just before the termination of the experiment at 5-wk of age or at an earlier time point. Therefore, it is important to interpret the data presented in this study with caution and keeping in mind that gnotobiotic pigs were monoassociated with C. perfringens and were not germ-free.

The staining patterns observed in this study showed that ileal tissues from gnotobiotic piglets, in general, had consistently weaker staining for various lectins such as LCA and PSA compared with the control and antibiotic piglets. These findings suggest that conventional intestinal microflora in pigs may be important in imparting changes on the glyoconjugate composition of the intestinal epithelial coat and Peyer’s patches (Umesaki et al., 1982Go; Umesaki, 1984Go; Wroblewski et al., 2001Go; Freitas et al., 2005Go). These observations are consistent with many previous findings described by others in germ-free mice and rat studies (Simon and Gorbach, 1984Go; Uribe et al., 1994Go; Sharma and Schumacher, 1995Go; Freitas et al., 2002Go). We observed that the absence of specific intestinal microflora in gnotobiotic pigs did not induce the overall downregulation of different carbohydrates in the ileum; rather, it was a selective process.

Significant differences in lectin binding between control or antibiotic vs. gnotobiotic pigs in the epithelial compartment (dome and villous epithelium) were observed mainly for RCA120, LEL, Jacalin, PSA, LCA, and SNA. Recent studies consistent with these observations showed that Bacterioides thetaiotaomicron, a member of intestinal microflora of mice and humans, specifically modified the galactosylation patterns of intestinal epithelium in vitro and in vivo (Freitas et al., 2001Go, 2005Go). It is important to note that in this study, not all lectins belonging to a specific sugar group displayed differences in lectin binding. This may be partly because each sugar moiety is linked to its backbone through a different linkage. Goblet cells showed weaker staining in gnotobiotic pigs for GSL-I, SBA, and PNA only. These observations are consistent with earlier mouse studies that showed the differential expression of carbohydrate moieties on the goblet cells of germ-free and conventional animals (Wroblewski et al., 2001Go; Freitas et al., 2005Go). None of the lectins that showed differences in binding on the dome and villous epithelium showed differences on goblet cells in gnotobiotic pigs. These distinct lectin binding profiles of goblet cells compared with enterocytes might reflect the functional differences in these cells.

A different set of lectins, namely WGA, PSA, LCA, and MAL, showed weaker binding on dome, corona, and follicles in gnotobiotic pigs compared with control or antibiotic pigs, or both. These observations suggest that some lectins such as PSA and LCA (glucose/mannose-specific group) have consistently lower binding in gnotobiotic pigs on both epithelial surfaces and Peyer’s patches, indicating the specific effect of intestinal microbiota in controlling the expression of these carbohydrates in the pig ileum. These findings may have implications on the growth and susceptibility of gnotobiotic pigs to various infections because many of carbohydrate moieties are involved in the nutritional processes (Banwell et al., 1983Go; Vasconcelos and Oliveira, 2004Go) and pathogen adherence in the intestine (Mouricout, 1997Go; Karlsson, 1998Go; Dai et al., 2000Go). Another possibility is that expression of glucose/mannose glycoconjugates may be required for the maturation of the gut and normal immune responses (Radberg et al., 2001Go; van Nevel et al., 2003Go; Vasconcelos and Oliveira, 2004Go).

The increased intensity of staining observed in the control and antibiotic-fed animals and the increased weight gain in these animals compared with gnotobiotic animals is a clear indication of the necessity of intestinal microbes in modulating the sugar moieties for promoting body weight gain in these animals. Further studies should be performed in germ-free pigs monocontaminated with individual microbes before drawing conclusions regarding the function of each microbe in regulating the intestinal glycosylation pattern and growth performance of animals.


   Footnotes
 
1 Acknowledgments: The authors thank H. Stein, Department of Animal Science, South Dakota State University (SDSU), for providing experimental animals and formulating animal feed for the study. Support for this project was provided by the Center for Infectious Disease Research and Vaccinology (CIDRV) and the Agricultural Experiment Station (AES), SDSU. The authors also thank the Departments of Biology and Microbiology, and Veterinary Science, SDSU, for providing facilities for the experiments. Back

2 South Dakota Agricultural Experiment Station (AES) Journal series number 3581. Back

3 Present address: Institute of Biotechnology, Universit Victor Segalen Bordeaux 2, Bordeaux, France. Back

4 Corresponding author: radhey.kaushik{at}sdstate.edu

Received for publication September 29, 2006. Accepted for publication March 22, 2007.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


Arias, C. F., P. Isa, C. A. Guerrero, E. Mendez, S. Zarate, T. Lopez, R. Espinosa, P. Romero, and S. Lopez. 2002. Molecular biology of rotavirus cell entry. Arch. Med. Res. 33:356–361.[CrossRef][Medline]

Atwood, C. S., and P. E. Hartmann. 1992. Collection of fore and hind milk from the sow and the changes in milk composition during suckling. J. Dairy Res. 59:287–298.[Medline]

Baker, D. R., R. A. Moxley, and D. H. Francis. 1997. Variation in virulence in the gnotobiotic pig model of O157:H7 Escherichia coli strains of bovine and human origin. Adv. Exp. Med. Biol. 412:53–58.[Medline]

Banwell, J. G., D. H. Boldt, J. Meyers, and F. L. Weber Jr. 1983. Phytohemagglutinin derived from red kidney bean (Phaseolus vulgaris): A cause for intestinal malabsorption associated with bacterial overgrowth in the rat. Gastroenterology 84:506–515.[Medline]

Brander, G. C. 1999. EU ban on four antibiotic feed additives. Vet. Rec. 144:104.[Medline]

Brown, P. J., S. S. Poulsen, and M. Wells. 1991. Serine protease immunohistochemistry and lectin histochemistry in the small intestine of weaned and unweaned pigs. Res. Vet. Sci. 50:1–5.[Medline]

Butler, J. E., D. H. Francis, J. Freeling, P. Weber, and A. M. Krieg. 2005. Antibody repertoire development in fetal and neonatal piglets. IX. Three pathogen-associated molecular patterns act synergistically to allow germfree piglets to respond to type 2 thymus-independent and thymus-dependent antigens. J. Immunol. 175:6772–6785.[Abstract/Free Full Text]

Butler, J. E., P. Weber, M. Sinkora, D. Baker, A. Schoenherr, B. Mayer, and D. Francis. 2002. Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens. J. Immunol. 169:6822–6830.[Abstract/Free Full Text]

Chae, C. 1997. Lectin histochemical characteristics of the epithelial surface of ileal Peyer’s patches in 3-week-old pigs. J. Vet. Med. Sci. 59:931–934.[CrossRef][Medline]

Chae, C., and Y. S. Lee. 1995. Age-related lectin histochemical changes in the porcine small intestine. J. Vet. Med. Sci. 57:883–889.[Medline]

Choi, B. Y., Y. S. Sohn, C. Choi, and C. Chae. 2003. Lectin histochemistry for glycoconjugates in the small intestines of piglets naturally infected with Isospora suis.J. Vet. Med. Sci. 65:389–392.[CrossRef][Medline]

Cromwell, G. L. 2002. Why and how antibiotics are used in swine production. Anim. Biotechnol. 13:7–27.[CrossRef][Medline]

Dai, D., N. N. Nanthkumar, D. S. Newburg, and W. A. Walker. 2000. Role of oligosaccharides and glycoconjugates in intestinal host defense. J. Pediatr. Gastroenterol. Nutr. 30(Suppl 2):S23–S33.[CrossRef][Medline]

Dormitzer, P. R., Z. Y. Sun, G. Wagner, and S. C. Harrison. 2002. The rhesus rotavirus vp4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site. EMBO J. 21:885–897.[CrossRef][Medline]

Farthing, M. J. 1985. Receptors and recognition mechanisms in intestinal infection. Trans. R. Soc. Trop. Med. Hyg. 79:569–576.[CrossRef][Medline]

Freitas, M., L. G. Axelsson, C. Cayuela, T. Midtvedt, and G. Trugnan. 2002. Microbial-host interactions specifically control the glycosylation pattern in intestinal mouse mucosa. Histochem. Cell Biol. 118:149–161.[Medline]

Freitas, M., L. G. Axelsson, C. Cayuela, T. Midtvedt, and G. Trugnan. 2005. Indigenous microbes and their soluble factors differentially modulate intestinal glycosylation steps in vivo. Use of a "lectin assay" to survey in vivo glycosylation changes. Histochem. Cell Biol. 124:423–433.[CrossRef][Medline]

Freitas, M., C. Cayuela, J. M. Antoine, F. Piller, C. Sapin, and G. Trugnan. 2001. A heat labile soluble factor from Bacteroides thetaiotaomicron vpi-5482 specifically increases the galactosylation pattern of ht29-mtx cells. Cell. Microbiol. 3:289–300.[CrossRef][Medline]

Freter, R. 1981. Mechanisms of association of bacteria with mucosal surfaces. Ciba Found. Symp. 80:36–55.[Medline]

Gaskins, H. R., C. T. Collier, and D. B. Anderson. 2002. Antibiotics as growth promotants: Mode of action. Anim. Biotechnol. 13:29–42.[CrossRef][Medline]

Gelberg, H., H. Whiteley, G. Ballard, J. Scott, and M. Kuhlenschmidt. 1992. Temporal lectin histochemical characterization of porcine small intestine. Am. J. Vet. Res. 53:1873–1880.[Medline]

Grange, P. A., A. K. Erickson, T. J. Anderson, and D. H. Francis. 1998. Characterization of the carbohydrate moiety of intestinal mucin-type sialoglycoprotein receptors for the k88ac fimbrial adhesin of Escherichia coli. Infect. Immun. 66:1613–1621.[Abstract/Free Full Text]

Grange, P. A., A. K. Erickson, S. B. Levery, and D. H. Francis. 1999. Identification of an intestinal neutral glycosphingolipid as a phenotype-specific receptor for the k88ad fimbrial adhesin of Escherichia coli. Infect. Immun. 67:165–172.[Abstract/Free Full Text]

Grange, P., B. Vedrine, and M. Mouricout. 1997. Adhesion of k88ab fimbriated E. coli in piglet small intestines in relation with iron transport molecules. Adv. Exp. Med. Biol. 412:357–361.[Medline]

Hathaway, M. R., W. R. Dayton, M. E. White, T. L. Henderson, and T. B. Henningson. 1996. Serum insulin-like growth factor 1 (IGF-1) concentrations are increased in pigs fed antimicrobials. J. Anim. Sci. 74:1541–1547.[Abstract]

Jaeger, L. A., C. H. Lamar, and J. J. Turek. 1989. Lectin binding to small intestinal goblet cells of newborn, suckling, and weaned pigs. Am. J. Vet. Res. 50:1984–1987.[Medline]

Jeong, K. I., Y. S. Sohn, K. Ahn, C. Choi, D. U. Han, and C. Chae. 2002. Lectin histochemistry of Peyer’s patches in the porcine ileum. J. Vet. Med. Sci. 64:535–538.[CrossRef][Medline]

Karlsson, K. A. 1998. Meaning and therapeutic potential of microbial recognition of host glycoconjugates. Mol. Microbiol. 29:1–11.[CrossRef][Medline]

Kelly, P., D. L. Jack, A. Naeem, B. Mandanda, R. C. Pollok, N. J. Klein, M. W. Turner, and M. J. Farthing. 2000. Mannose-binding lectin is a component of innate mucosal defense against Cryptosporidium parvum in AIDS. Gastroenterology 119:1236–1242.[CrossRef][Medline]

Klobasa, F., E. Werhahn, and J. E. Butler. 1987. Composition of sow milk during lactation. J. Anim. Sci. 64:1458–1466.[Abstract/Free Full Text]

Ley, R. E., F. Backhed, P. Turnbaugh, C. A. Lozupone, R. D. Knight, and J. I. Gordon. 2005. Obesity alters gut microbial ecology. Proc. Natl. Acad. Sci. USA 102:11070–11075.[Abstract/Free Full Text]

Mendez, E., S. Lopez, M. A. Cuadras, P. Romero, and C. F. Arias. 1999. Entry of rotaviruses is a multistep process. Virology 263:450–459.[CrossRef][Medline]

Mouricout, M. 1997. Interactions between the enteric pathogen and the host. An assortment of bacterial lectins and a set of glycoconjugate receptors. Adv. Exp. Med. Biol. 412:109–123.[Medline]

Ozawa, E. 1955. Studies on growth promotion by antibiotics. I. Effects of chlortetracycline on growth. J. Antibiot. (Tokyo) 8:205–211.[Medline]

Pusztai, A. 1996. Characteristics and consequences of interactions of lectins with the intestinal mucosa. Arch. Latinoam. Nutr. 44:10S–15S.[Medline]

Pusztai, A., S. W. Ewen, G. Grant, D. S. Brown, J. C. Stewart, W. J. Peumans, E. J. Van Damme, and S. Bardocz. 1993. Antinutritive effects of wheat-germ agglutinin and other n-acetylglucosamine-specific lectins. Br. J. Nutr. 70:313–321.[CrossRef][Medline]

Radberg, K., M. Biernat, A. Linderoth, R. Zabielski, S. G. Pierzynowski, and B. R. Westrom. 2001. Enteral exposure to crude red kidney bean lectin induces maturation of the gut in suckling pigs. J. Anim. Sci. 79:2669–2678.[Abstract/Free Full Text]

Schwarz, S., and E. Chaslus-Dancla. 2001. Use of antimicrobials in veterinary medicine and mechanisms of resistance. Vet. Res. 32:201–225.[CrossRef][Medline]

Sharma, R., and U. Schumacher. 1995. The influence of diets and gut microflora on lectin binding patterns of intestinal mucins in rats. Lab. Invest. 73:558–564.[Medline]

Sharon, N. 1987. Bacterial lectins, cell-cell recognition and infectious disease. FEBS Lett. 217:145–157.[CrossRef][Medline]

Shirkey, T. W., R. H. Siggers, B. G. Goldade, J. K. Marshall, M. D. Drew, B. Laarveld, and A. G. Van Kessel. 2006. Effects of commensal bacteria on intestinal morphology and expression of proinflammatory cytokines in the gnotobiotic pig. Exp. Biol. Med. (Maywood) 231:1333–1345.[Abstract/Free Full Text]

Simon, G. L., and S. L. Gorbach. 1984. Intestinal flora in health and disease. Gastroenterology 86:174–193.[Medline]

Sohn, Y. S., and C. Chae. 2000. Lectin-binding capacity of glycoconjugates in Escherichia coli 09:K103:Nm, 987P+ST+-infected porcine lower small intestines. J. Vet. Med. Sci. 62:543–547.[CrossRef][Medline]

Sousa, M. C., C. A. Goncalves, V. A. Bairos, and J. Poiares-Da-Silva. 2001. Adherence of Giardia lamblia trophozoites to INT-407 human intestinal cells. Clin. Diagn. Lab. Immunol. 8:258–265.[CrossRef][Medline]

Stanley, S. L., Jr., and S. L. Reed. 2001. Microbes and microbial toxins: Paradigms for microbial-mucosal interactions. VI. Entamoeba histolytica: Parasite-host interactions. Am. J. Physiol. Gastrointest. Liver Physiol. 280:G1049–G1054.[Abstract/Free Full Text]

Stewart, J. A., V. S. Chadwick, and A. Murray. 2005. Investigations into the influence of host genetics on the predominant eubacteria in the faecal microflora of children. J. Med. Microbiol. 54:1239–1242.[Abstract/Free Full Text]

Tavares, P., M. C. Rigothier, H. Khun, P. Roux, M. Huerre, and N. Guillen. 2005. Roles of cell adhesion and cytoskeleton activity in Entamoeba histolytica pathogenesis: A delicate balance. Infect. Immun. 73:1771–1778.[Abstract/Free Full Text]

Umesaki, Y. 1984. Immunohistochemical and biochemical demonstration of the change in glycolipid composition of the intestinal epithelial cell surface in mice in relation to epithelial cell differentiation and bacterial association. J. Histochem. Cytochem. 32:299–304.[Abstract]

Umesaki, Y., K. Tohyama, and M. Mutai. 1982. Biosynthesis of microvillus membrane-associated glycoproteins of small intestinal epithelial cells in germ-free and conventionalized mice. J. Biochem. (Tokyo) 92:373–379.[Abstract/Free Full Text]

Uribe, A., M. Alam, O. Johansson, T. Midtvedt, and E. Theodorsson. 1994. Microflora modulates endocrine cells in the gastrointestinal mucosa of the rat. Gastroenterology 107:1259–1269.[Medline]

Vaahtovuo, J., P. Toivanen, and E. Eerola. 2003. Bacterial composition of murine fecal microflora is indigenous and genetically guided. FEMS Microbiol. Ecol. 44:131–136.[CrossRef]

van Nevel, C. J., J. A. Decuypere, N. Dierick, and K. Molly. 2003. The influence of Lentinus edodes (shiitake mushroom) preparations on bacteriological and morphological aspects of the small intestine in piglets. Arch. Tierernahr. 57:399–412.[Medline]

Vasconcelos, I. M., and J. T. Oliveira. 2004. Antinutritional properties of plant lectins. Toxicon 44:385–403.[Medline]

Wolter, B. F., M. Ellis, B. P. Corrigan, and J. M. DeDecker. 2002. The effect of birth weight and feeding of supplemental milk replacer to piglets during lactation on preweaning and postweaning growth performance and carcass characteristics. J. Anim. Sci. 80:301–308.[Abstract/Free Full Text]

Wroblewski, S., B. Rihova, P. Rossmann, T. Hudcovicz, Z. Rehakova, P. Kopeckova, and J. Kopecek. 2001. The influence of a colonic microbiota on hpma copolymer lectin conjugates binding in rodent intestine. J. Drug Target. 9:85–94.[Medline]

Zijlstra, R. T., K. Y. Whang, R. A. Easter, and J. Odle. 1996. Effect of feeding a milk replacer to early-weaned pigs on growth, body composition, and small intestinal morphology, compared with suckled littermates. J. Anim. Sci. 74:2948–2959.[Abstract]



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Figure 1
Right arrow All Versions of this Article:
jas.2006-662v1
85/7/1640    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by George, S.
Right arrow Articles by Kaushik, R. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by George, S.
Right arrow Articles by Kaushik, R. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS