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Keywords:

  • antimicrobial peptides;
  • enteritis;
  • fish;
  • intestinal segments;
  • mucins

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The alimentary tract is a possible site where pathogens and toxins can enter. The alimentary tract is protected, amongst others, by mucus. In this study, tissue samples and crude mucus preparations from different parts of the intestinal tract of Cyprinus carpio (from intestinal bulb onto the hindgut) were examined using histological, histochemical and biochemical techniques. Furthermore, the response of the intestinal mucosal layer and intestinal mucus of C. carpio to acute soybean meal (SBM)-induced enteritis was investigated. In the present study, an indication for a different protein core of mucus high molecular weight glycoproteins (HMGs) for first and second segment could not be found. However, differences in mucus glycosylation could be found. Along the gut axis, the size of the major protein peaks were not similar, which can be caused by a different glycosylation. Also, differences in staining for the antimicrobial peptide beta-defensin 2 were found. Furthermore, changes in HMGs upon SBM diet were found similar to those found in inflammatory bowel disease (IBD) in humans. Initial changes include: changes in mucin composition, the presence of BD3 and of bacteria in internal organs. After the initial changes, all values measured returned back to the initial pre-SBM diet values.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Barrier function of the alimentary tract

The main function of the alimentary tract of fish as well as any other vertebrates is the acquisition of food with subsequent assimilation of vital nutrients and defence against pathogens (Bakke et al. 2010). In fish, the alimentary tract also plays a role in osmoregulation (Olsson 2011). The intestinal barrier is formed by extrinsic, intrinsic and immunological barriers. The first-barrier pathogens and toxins encounter is the extrinsic barrier. This barrier is mainly formed by mucus that counters pathogens and toxins. The intrinsic barrier consists of epithelial cells and cell junctions. Molecules and cells of the innate and the adaptive immune system that are present in the intestinal wall form the immunological barriers.

The extrinsic barrier mucus consists mostly of two components that are both important in gel formation (Verdugo 1990; Strous & Dekker 1992; Bansil et al. 1995; Cone 1999; Perez-Vilar & Hill 1999). These components are water and high molecular weight glycoproteins (HMGs), called ‘mucins’. Mucins exhibit a high content of oligosaccharides and form a water-insoluble layer. Preventing pathogens from penetrating the mucus layer relies not only on mucus flow and mucin composition, but also on other mucus components such as antimicrobial peptides or lysozyme. Knowledge about fish mucus and especially about its HMGs is still scarce. In addition, the composition of the commensal microbiota and therefore also of potential pathogens in fish is still limited.

The gastrointestinal tract of fish can be subdivided into four topographical regions: the headgut, foregut, midgut and hindgut (Harder 1975). The headgut is composed of the mouth and pharynx, and the foregut is formed by oesophagus and stomach (Clements & Raubenheimer 2005). In carp as an agastric fish, the foregut only comprises the oesophagus. The midgut or intestine accounts for the greatest proportion of the gut length, and here, the chemical digestion of ingested food is continued and absorption of nutrients mainly occurs. In carp, the anterior intestine bulges to form an intestinal bulb, which functions in temporary food storage; however, gastric glands and a pylorus are lacking. The hindgut is the final section of the gut, which includes the rectum (Wilson & Castro 2010).

In the absorbing parts of the gut, there are differences in barrier function between regions. In teleosts, the anterior intestine including the pyloric ceca, which are present in some species, are the main nutrient-absorbing regions. The lumen in this region contains high nutrient concentrations, typically relative low numbers of bacteria, and is lined by an epithelium of high paracellular permeability. In the posterior intestine, however, bacterial numbers are higher and free nutrient concentrations drop. As the nutrient content drops along the intestinal tract and bacteria and bacterial toxins rise in concentration, the need for a tighter barrier increases (Jutfelt 2011). Therefore, in the absorbing parts of the gut, differences in the barrier function between regions are found.

Soybean meal and enteritis

Optimal feeding of fish has been the subject of extensive study. As, in aquaculture, feed costs often comprise more than 50% of total production costs (El-Sayed 1999; Fagbenro 1999), a cheap and reliable source of protein is needed for both economic and sustainability reasons. Soybean Glycine max L. has a great potential as protein and/or oil source for fish feed (Alexis & Nengas 2001). However, soybean meal (SBM) containing diets are known to have the potential to induce an inflammatory response in the hindgut of certain fish species. So far, most studies have been conducted on Salmo salar and have mainly focused on the effect of SBM-containing diets on intestinal morphology, growth, enzyme activity and metabolism. A negative influence was observed for these parameters (Corfield et al. 1993; Baeverfjord & Krogdahl 1996; Bakke-McKellep et al. 2000, 2007; Krogdahl et al. 2003) and on disease resistance (Krogdahl et al. 2000) as well as immune relevant genes (Lilleeng et al. 2009). In common carp Cyprinus carpio L., which were fed a diet where 20% of the protein was replaced by SBM, enteritis was induced in the hindgut (Uran et al. 2008). Contrary to previous observations made with S. salar, C. carpio start to recover or adapt to the SBM feed from the fourth week after the SBM feeding.

In humans, increased gut permeability has been described following enteritis. If changes after enteritis persist, a chronic inflammation can develop (Dunlop et al. 2006). Chronic inflammatory bowel diseases (IBD) are well described for humans. In IBD, genetic mutations in mucin genes, changes in sulphation, degree of glycosylation, mucin mRNA, protein levels and degradation of mucins have been described (Einerhand et al. 2002). In the research on SBM-induced enteritis in fish, no attention has been paid so far to the role of mucins.

The present study

In C. carpio, the presence of intestinal glycoproteins that are similar in structure and composition to those found in mammals has been documented (Neuhaus et al. 2007b), but until now, data did not include a differentiation of functional intestinal segments based on mucus composition. In this study, tissue samples and crude mucus preparations from different parts of the intestinal tract of C. carpio (from intestinal bulb onto the hindgut) were examined using histological, histochemical and biochemical techniques. A distinction was made between non-secreted mucus HMGs present in the goblet cells and secreted mucus HMGs present in the lumen. Furthermore, in this study, the response of the intestinal mucosal layer and of intestinal mucus of C. carpio to acute SBM-induced enteritis was examined.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Fish and rearing conditions

Clinically healthy, parasite and virus–free C. carpio from a single crossing (Wageningen Agricultural University, Netherlands) were used throughout the study. From day 4, posthatching until 4 weeks of age, larvae were fed Artemia salina nauplii. To examine possible differences in mucin composition between intestinal segments, C. carpio (120 ± 21 g, strain E20 × R8) that had been fed with commercial trout pellets (Trouvit) were used.

To examine a possible effect of SBM on mucin composition, C. carpio were transferred to another system and kept in recirculating, filtered, UV-sterilized water at 23 °C. Cyprinus carpio were then fed a fishmeal-based diet without soybean protein (0SBM). For experimentation, fish were switched to an experimental diet (20SBM), in which fish meal, fish oil and wheat were exchanged for 20% SBM. Feed was produced as extruded sinking pellets (Skretting, Aquaculture Research Centre, Stavanger, Norway) and were formulated to be iso-nitrogenous and iso-energetic on a crude protein and a crude lipid basis. The fish (221.7 ± 47.2 g, stain R8 × R3) were manually fed 4% of their body weight per day, which was divided into two equal servings and given by hand. Both diets (0 SBM and 20 SBM) have been described earlier by Uran et al. (2008).

Sampling

Cyprinus carpio were starved for 2 days before sampling to reduce faecal contamination of the intestinal mucus. Cyprinus carpio were euthanized with 0.03% tricaine methane sulphonate (TMS; Crescent Research Chemicals, Phoenix, AZ, USA), buffered with 0.06% sodium bicarbonate to a pH of 7.2. The entire intestinal tract was removed, cooled on ice and divided into three parts: intestinal bulb, rest of the first segment and second segment. Small tissue pieces (3–4 mm) were taken from the intestinal segments for histology, and the remaining tissue was used for mucus isolation.

Fish were sampled at the start of the experiment at week 0 (n = 8) and weeks 1, 2 and 3. (at each of these time points n = 5). Furthermore, one bacterial swab was taken from internal organs (liver, kidney and spleen) from these fish for microbiological examination. Swabs were cultivated on blood agar plates for 2 days at 25 °C. Bacteria were identified with the API system (BioMerieux, Craponne, France). Differences to control were tested to one-way anova on Ranks.

Histology and immunohistochemistry

By means of histology and immunohistochemistry, the composition and amount of complex carbohydrates within the mucus in the goblet cells as well as the presence of antimicrobial substances were examined. Hereto, fresh intestinal tissue was immersed in Carnoy's fluid (staining for complex carbohydrates, segment analysis samples) (Ota & Katsuyama 1992) or Bouin's fluid. Samples were immersed in Bouin's fluid for at least 4 days at room temperature. Samples were immersed in Carnoy's for 2 h at room temperature and then transferred to, and washed in isopropanol. All samples were then washed, dehydrated in a series of graded ethanol and embedded in paraffin wax. Subsequently, 5 μm paraffin sections were cut.

Mucin carbohydrates were visualized with periodic-acid Schiff (PAS), Alcian Blue 8GX pH 1.0 (AB1.0) as well as a AB2.5 (segment analysis samples) or AB2.5/Crossmann (0 SBM and 20 SBM) (Romeis 1968). The PAS reaction visualizes neutral glycoconjugates; the AB1.0 method stains sulphated glycoconjugates; the AB2.5 method stains acidic glycoconjugates. All sections were studied or photographed with a light microscope (Zeiss light microscope Axiphot; Zeiss, Oberkochem, Germany).

The presence of antimicrobial substances [ß-defensin 2 (BD2), ß-defensin 3 (BD3) and lysozyme-muramidase (Lys)] was examined by immunohistochemical indirect visualization. Hereto, sections were deparaffinized in xylene and carefully hydrated through descending concentrations of ethanol. Sections for lysozyme staining were incubated for 30 min with 0.1% trypsin (pH 7.8, 37 °C) to demask the proteins. Subsequently, all sections were pretreated for 20 min with normal goat serum (NGS). Then, sections were incubated with the primary antibody, which was diluted with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The polyclonal primary antibody BD2 was diluted 1 : 500, BD3 1 : 1000 and Lys 1 : 100, and all three were obtained from rabbit immunized with human peptides (Biologo, Kronshagen, Germany). Following overnight incubation at 4 °C, the reaction was detected by the EnVision system (Dako, Hamburg, Germany). Control sections were incubated without the primary antibody to test for unspecific binding of the visualization substrate. Furthermore, sections were incubated without the primary, and without the secondary antibody, but with the visualization substrate to test for endogenous enzyme activity. Antibody specificity has been previously described (Hübner 2012).

The presence of antimicrobial substances in the goblet cells was estimated on an arbitrary scale (none, few, several, many) for both the number of folds with stained goblet cells and the number of stained goblet cells per fold. The carbohydrate stains were evaluated on the basis of the goblet cell reaction. The number of responding goblet cells was counted in four intestinal folds each over a fixed length (390 μm) of epithelial lining per fold. The mean and standard deviation of all measurements per fish were calculated. Furthermore, staining intensity of the goblet cells (faint, weak, moderate, strong, very strong) was evaluated on an arbitrary scale. The number of goblet cells was statistically analysed with a one-way anova. Goblet cell staining intensity was statistically analysed with a Kruskall–Wallis one-way anova on ranks. Differences were considered as significant at a probability of error of P ≤ 0.05.

Isolation of HMGs from intestinal mucus

For mucus isolation, the intestinal segments were opened longitudinally and cut into small pieces of 3–4 mm. Subsequently, secreted HMGs were isolated with isolation medium as described previously (Schroers et al. 2009). In brief, tissue pieces were incubated for 20 min in 100-mL isolation buffer containing antibiotics and protease inhibitors (Amphotericin B, dithiothreitol, sodium pyruvate, HEPES).

The isolation buffer was collected, centrifuged for 30 min at 12 000× g, and the supernatant was collected and frozen at −20 °C until further processing. Non-secreted mucus HMGs were released from goblet cells by subsequent incubation of the tissue pieces for 30 min in a buffer-containing EDTA, with the addition of the same antibiotics and protease inhibitors as in the isolation medium for secreted mucus. Goblet cells were disrupted by means of an ultrasonic unit (Ultra Turrax T8; IKA-Werke, Staufen, Germany). The suspension was centrifuged at 10 000 g for 30 min, and the supernatant was collected and homogenized by gentle stirring. All samples were concentrated by ultrafiltration (Amicon, Beverly, MA, USA, exclusion limit 30 kDa) to a final volume of 2 mL and used for size-exclusion chromatography.

High-performance liquid chromatography

Mucus samples for segment analysis were analysed for the presence of monosaccharides by reverse-phase high-performance liquid chromatography (HPLC) with the method described by Anumula (1994). In brief, the mucus samples were hydrolysed with 20% trifluoroacetic acids and then derivatized with an excess of anthranilic acid in the presence of sodium cyanoborohydride. Antranilic acid derivatives of monosaccharides were separated on a HPLC column using a 1-butylamine-phosphoric acid-tetrahydrofuran mobile phase. All separations were carried out at ambient temperature using a flow rate of 1 mL min−1. Solvent A consisted of 0.25% 1-butylamine, 0.5% phosphoric acid, 1% tetrahydrofuran (inhibited) in water. Solvent B consisted of equal parts of solvent A and acetonitrile. The elution programme was 5% of solvent B for 25 min followed by a linear increase to 15% of solvent B at 50 min. The column was washed for 15 min with 100% of solvent B and equilibrated for 15 min with 5% of solvent B. Elution was monitored by measuring the fluorescence (excitation wavelength, 230 nm; emission wavelength, 425 nm).

Sugars were identified by comparing retention times (RT) of peaks in the samples with RT of peaks of standard sugar solutions. Standard sugar solutions were galactose (gal) RT 12.1 min, N-acetylneuraminic acid (NeuNAc) RT 12.6 min, glucose (glc) RT 12.8 min, mannose (man) RT 13.2 min, fucose (fuc) RT 18.2 min and N-acetylgalactosamine (galNAc) RT 23.4 min. For each identified peak in the samples, surface areas of eluted peaks were compared with surface area of peaks of standard sugar solutions with a known amount of monosaccharide. Total sugar quantities and the monosaccharide percentage of the total sugar quantities were calculated. Data were analysed with a t-test. Differences between intestinal segments and differences between secreted and non-secreted mucus of the same segment were considered as significant at a P value of ≤0.05.

Size-exclusion chromatography of mucus samples

Size-exclusion chromatography was performed on an ÄktaFPLC-System (GE Healthcare, Freiburg, Germany). Sepharose CL-4B (Sigma-Aldrich, Munich, Germany) was used in an XK 16/40 column (GE Healthcare). PBS at pH 7.2 was used as running buffer. Up to 2 mL of concentrated mucus samples were loaded on the column. In a steady buffer stream of 1 mL min−1, the proteins in the mucus concentrate were separated by size. Elution of the proteins was monitored online via UV activity (280 nm) and conductivity.

To determine the molecular weight of the eluted proteins, solutions with known molecular weights were also monitored. Hereto, apoprotein (8 kDa), FK506-binding protein (12 kDa), MBP2* protein (40 kDa) and MBP-β-galactosidase (158 kDa) were subjected to size-exclusion chromatography (Fig. 1). Differences were statistically evaluated with an anova on Ranks (P < 0.05).

image

Figure 1. Goblet cell staining at the fold base for BD2. Shown are sections of the first (left) and second (right) intestinal segment of carp fed with standard trout feed.

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Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Histology

Goblet cells with sulphated, acidic and neutral glycoproteins were found in all gut regions. In all stainings and all intestinal segments, the goblet cells had an oval to rounded form. For carp that were kept on normal trout feed, a significant difference in goblet cell number and staining intensity could be found for some stainings between the intestinal segments (Table 1). Goblet cells in the second segment showed a significantly weaker staining than in the first segment for the AB2.5 and PAS stain (acidic and neutral glycoconjugates, respectively). The number of goblet cells that were stained in the intestinal bulb was significantly lower than the number in the first and second segment for AB1.0 and PAS. The number of AB2.5-stained goblet cells was also lower in the intestinal bulb, but the differences were not significant.

Table 1. Number of stained goblet cells (mean ± SD) per 390 μm of epithelial lining per fold and median goblet cell staining intensity of three intestinal segments after staining for complex carbohydrates
 Intestinal bulb1st segment2nd segment
  1. Different superscripts indicate significant differences between intestinal segments (one-way anova, P ≤ 0.05).

  2. PAS, periodic-acid Schiff.

Goblet cell number
AB1.014.5 ± 3.5a20.4 ± 3.4b18.6 ± 3.0b
AB2.515.0 ± 3.819.2 ± 2.118.0 ± 3.5
PAS14.5 ± 3.5a20.4 ± 3.4b18.6 ± 3.0b
Staining intensity
AB1.0StrongStrongStrong
AB2.5StrongabStrongaModerateb
PASStrongabVery strongaStrongb

After switching from the 0 SBM to the 20 SBM diet, significant differences between the 0 SBM and 20 SBM fish could be found for some carbohydrate stainings in the goblet cell number (Table 2). For the sulphated glycoconjugates (AB1.0), the number of stained goblet cells was significantly lower on week 2 post diet change and significantly higher the week after. For the acidic glycoconjugates (AB2.5), significant differences were observed in the second intestinal segment. Here, the goblet cell number was significantly lower at week 1 after switching to 20 SBM and significantly higher at week 3 after switching to 20 SBM. For the neutral glycoconjugates (PAS), significant differences could be found for both intestinal segments for all sampling points compared with the 0 SBM. Here, a significantly higher goblet cell number was found for the first intestinal segment and a significantly lower number for the second segment. No clear effect of switching to 20 SBM could be observed on the presence of supranuclear vacuoles.

Table 2. Per cent change in number of goblet cells compared with control after changing the diet from 0SBM to 20SBM
StainingIntestinal segmentTime point after diet change
Week 1Week 2Week 3
  1. a

    Values are averages ± standard deviation. Significant differences (one-way anova, P ≤ 0.05) to control are indicated with *.

  2. PAS, periodic-acid Schiff.

AB1.01st106 ± 20%68 ± 28%a131 ± 26a
2nd88 ± 3%93 ± 21%85 ± 9%
AB2.51st95 ± 6%92 ± 25%104 ± 4%
2nd59 ± 16%a96 ± 18%117 ± 17%a
PAS1st122 ± 14%121 ± 21%123 ± 14%
2nd42 ± 12%a64 ± 8%a74 ± 17%a

Immunohistochemistry

For carp that were kept on a normal feeding regime, goblet cells could be stained with the antibodies against BD2, BD3 and lysozyme humoral substances (Table 3). However, goblet cells were mostly stained at the base of the intestinal fold. In the first segment, only a few goblet cells were stained on the flank base. Although goblet cells were visible in flank and tips of the folds, they showed low levels of antibody binding. In addition, a binding to the mucus overlay and to apical parts of the enterocytes at the basis of the mucosal folds could be observed.

Table 3. Staining for the presence of antimicrobial peptides in the base and the flank base of intestinal folds of first and second segments of naïve carp. Number of intestinal folds in which goblet cells could be stained (folds stained) and the number of stained goblet cells per stained fold (goblet cells stained) are indicated. Shown are median (min – max)
Humoral substancesPresence of staining1st segment2nd segment
  1. a

    Indicates significant differences between first and second segment.

BD2Folds stained of:
BaseMany (few – many)Few (none – several)a
Flank baseFew (none – few)None (none – none)a
Goblet cells stained in:
BaseSeveral (few – several)Few (none – few)a
Flank baseFew (none – few)None (none – none)
BD3Folds stained of:
BaseFew (few – several)Few (none – several)
Flank baseNone (few – none)None (none – none)
Goblet cells stained in:
BaseFew (few – several)Few (none – few)
Flank baseNone (none – few)None (none – none)
LysFolds stained of:
BaseNone (none – few)None (none – few)
Flank baseNone (none – none)None (none – none)
Goblet cells stained in:
BaseNone (none – few)None (none – few)
Flank baseNone (none – none)None (none – none)

For lysozyme and BD3, a few goblet cells per fold, and only a few folds with stained goblet cells could be observed at the base of the intestinal folds for both first and second segments. Only for BD2 could significant differences be observed between the first and second segment (Table 3, Fig. 1). The number of goblet cells per fold, which could be stained was significantly less in the second segment when compared with the first segment, and they were present in both base and flank base. The number of folds (base) with stained goblet cells was significantly lower in the second segment compared with the first segment.

After switching from the 0 SBM to the 20 SBM diet, significant differences to 0 SBM could only be observed for BD3. These differences were observed only at the fold base between the control and week 1 fish post diet change (Fig. 2.). The number of folds with stained goblet cells was significantly lower for the second intestinal segment at week 1 [none (none – few)] compared with the control [few (few – several)]. The number of goblet cells per 390 μm of epithelial lining per fold was also significantly lower at week 1 compared with control. This was found for both first intestinal segment [none (none – none) compared with several (few – several)] and second intestinal segment [none (none – few) compared with few (few – several)].

image

Figure 2. Goblet cell staining for BD3. Shown are sections of the first (left) and second (right) intestinal segment of 0SBM (top) and day 7 after switching to 20SBM samples (bottom).

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HPLC

Mucus samples of naïve fish were analysed for the presence of monosaccharides by reverse-phase HPLC (Table 4). Unfortunatly, due to similar RT, no clear separation could be made between NeuNAc and gal or between glc and man. In samples from the first segment, NeuNAc/gal was abundantly present. For non-secreted mucus, the percentage of NeuNAc/gal was significantly lower in the second segment when compared with first segment. For the second segment, glc/man and fuc could be frequently detected. GalNac could not be detected in the second segment. Therefore, the percentage of glc/man was significantly lower in the first than in the second segment for secreted mucus and for fuc in non-secreted mucus. One peak at RT 16 min could not be identified with the standard sugar solutions used. The peak area was large for both non-secreted and secreted mucus in the first segment (1.7 and 2.5 times the area of NeuNAc/gal). The area of the unidentified peak was smaller for the second segment. The size area was approximately that of the glc/man peak for the non-secreted mucus, and for secreted mucus, it was 1.2 times the size area of the glc/man peak.

Table 4. The presence of N-acetylneuraminic acid/galactose (NeuNAc/gal), glucose/mannose (glc/man), fucose (fuc) and N -acetylgalactosamine (galNAc) in non-secreted and secreted mucus from first segment (1-ns and 1-s, respectively) and second segment (2-ns and 2-s, respectively) of naïve carp
 1-ns1-s2-ns2-sSignificant differences (P < 0.05)
Total amount (μg)% of total16.0 ± 6.316.8 ± 6.12.4 ± 0.19.5 ± 0.22-ns < 2-s
NeuNAc/gal42.7 ± 5.739.0 ± 5.013.9 ± 2.627.7 ± 3.42-ns < 1-ns
glc/man22.6 ± 6.829.3 ± 5.332.0 ± 4.647.0 ± 2.41-s < 2-s
fuc15.4 ± 5.015.1 ± 4.754.2 ± 7.225.4 ± 1.01-ns < 2-ns
galNAc19.3 ± 6.616.7 ± 4.70.0 ± 0.00.0 ± 0.02-s < 1-s, 2-ns < 1-ns

Size-exclusion chromatography

In all examined samples, a biphasic profile could be observed. All samples showed a major protein residue between 120 and 180 kDa (Fig. 3) peaking at approximately 150 kDa. In all samples, a second major protein residue with molecules ranging between 12 and 70 kDa could be observed. The second major protein residue showed a high variation. In all residues, a major peak at approximately 40 kDa with a second flanking peak at approximately 25 kDa was observed. Although in all samples, the peak at approximately 40 and 25 kDa were visible, areas of the peaks were highly variable. Samples from the second intestinal segment showed a third peak in the second major residue at approximately 70 kDa. This peak was quite small with an average absorbence of 10 mAU, but it was still very prominent (Fig. 3).

image

Figure 3. Size exclusion chromatograms of non-secreted mucus (top) and secreted mucus (bottom) from the different intestinal segments of Cyprinus carpio. Indicated are standards for size exclusion chromatography with apoprotein (8 kDa), FK506 binding protein (12 kDa), MBP2* protein (40 kDa) and MBP-β-galactosidase (158 kDa).

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Size areas of the major protein residue between 120 and 180 kD were also highly variable. However, this major protein residue is a single peak with little variation in retention time. Between non-secreted and secreted mucus samples from the same intestinal segment, the retention time of the first peak was not significantly different. This was also true when samples from the first and second intestinal segment (peak at approximately 150 kDa) were compared. The protein peak from the mucus of the intestinal bulb had a slightly larger retention volume, indicating a slightly smaller protein size compared with the protein peaks from mucus of the first and second segments. Although size difference was small (approximately 10 kDa), the first major protein residue from the intestinal bulb had a significantly higher retention time compared with that of the first and second intestinal segment (P < 0.001).

All samples from the SBM experiment also showed a biphasic protein profile with a major protein peak at approximately 150 kDa. For non-secreted mucus in the first intestinal segment, switching to the 20 SBM diet lead to a significant increase in peak height (8.0-fold increase on day 7 compared with day 0). The increase became less over time [7.2-fold (significant) on day 14 and 5.3 fold on day 20]. No significant differences could be found for the peak height of the secreted mucus from the first intestinal segment and of both non-secreted and secreted mucus from the second intestinal segment.

Microbiological examination from the soybean experiment

None of the fish died during the soybean feeding experiment. From the 23 fish that were sampled from only seven, no bacteria could be cultured on blood agar plates. From all other carp facultative pathogenic bacteria (fluorescent and non-fluorescent Pseudomonas spp., Aeromonas sobria, Aeromonas veronii and Cytophaga-like bacteria) could be isolated.

Bacterial content in the organs was for most fish (twelve of sixteen) where bacteria could be isolated, low to moderate (Table 5). From none of the control fish or of the fish sampled 3 weeks after feed alteration, bacteria could be isolated in high numbers. On week 1 after feed alteration, significant higher bacteria intensity could be observed.

Table 5. Number of carp with a no, low, moderate and high bacterial content isolated from carp organs after switching to a 20 SBM diet
Bacterial contentTime point after diet change
Control (n = 8)Week 1a (n = 5)Week 2 (n = 5)Week 3 (n = 5)
  1. a

    Significant difference to control (P < 0 .05). Prevalence was compared with control and tested with one-way anova.

No2023
Low3011
Moderate3211
High0310

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

HMGs and antimirobial peptides in intestinal mucus

On fish mucin molecules, relatively few studies have been conducted, however, the data available indicate that fish mucus is similar in composition to the mucins secreted by mammalian goblet cells from epithelial tissues (Fletcher & Grant 1968; Asakawa 1974; Wold & Selset 1977; Lumsden & Ferguson 1994; Neuhaus et al. 2007b). For C. carpio, mucus glycoproteins have so far been described under physiological circumstances and after bacterial challenge for C. carpio (Neuhaus et al. 2007a,b; Schroers et al. 2009; Van der Marel et al. 2010). For mammals, it is well known that different parts of the gastrointestinal tract have different mucus thicknesses (Szentkuti & Lorenz 1995; Atuma et al. 2001) and express different mucus genes (Gendler & Spicer 1995; Van Klinken et al. 1995). For carp, so far two mucin genes have been found of which only one, Muc2, was expressed in the intestinal tract (Van der Marel et al. 2012). As only one Muc gene has been identified in the intestinal tact, it is not known whether different Muc gene profiles exist along the gut axis. However, morphological and functional differences have been previously found along the gut axis. Whether these differences between different parts of intestinal tract also include differences in mucus HMGs has to our knowledge not been extensively examined so far.

In this study, mucus glycoproteins within the goblet cells appeared to have a similar charge due to their glycosylation, as differences between the applied stainings could not be found. However, HPLC differences in the monosaccharide content of the mucus HMGs could be observed for both the first and second segments. Based on its molecular weight, the unidentified sugar in this study is probably ribose (Marko-Varga 1987). Ribose and 2-deoxyribose were the predominant sugars in rainbow trout intestinal mucus. For the first segment, the size area of the ‘ribose-peak’ was the highest. Fucose that is thought to be especially important for the viscoelasticity of the mucus, may vary strongly in intestinal mucus or anal gland mucus of mammals (Tsukise et al. 2000). The fucose content of C. carpio intestinal mucus was dependent on the origin of the mucus (first or second segment). For non-secreted mucus in the second segment, fucose was the most predominant sugar, as was also observed for C. carpio skin mucus (unpublished data). Non-secreted mucus is thought to be present in a native form, whereas secreted mucus might be altered by environmental influences such as bacterial degradation. In the first segment, NeuNAc/gal was predominant. The sugar galNAc could not be detected with the method used in the second segment, which was also the case for C. carpio skin mucus (unpublished data). In mammals, the monosaccharide profile of mucins is largely genetically determined (c.f. Freitas et al. 2005), however, can be modulated in response to bacterial colonization (Freitas et al. 2005). Research on germ-free animals indicated that the glycoproteins do not only play a role in binding of adverse molecules such as hormones, enzymes or microbial toxins, but also serve as nutrients for the commensal microbial flora. In germ-free mice, for instance, the expression of NeuNac was increased after oral application of bacteria from the conventional microflora, while the expression of GlycNac was decreased (Freitas et al. 2005). A high expression of Gal1,3Gal at the portal to the zebrafish intestine was considered to have a function in the transient recruitment of gut mutualists. Aeromonads members from the zebrafish gut flora preferentially bound to this sugar when expressed on cells. (Cheesman & Guillemin 2007). This might explain the high expression of GalNac in the first segment (recruitment of gut flora) and its decreased expression in the second segment. In general, on the monosaccharide level, C. carpio skin mucus resembled the mucus of the second segment more than the mucus of the first segment.

Besides sugar analysis, protein content was examined by size-exclusion chromatography. In all examined samples, a biphasic profile could be observed. Non-secreted and secreted mucus samples from the different intestinal segments contained almost similar protein profiles: their residues are between 120–180 kDa and 12–70 kDa. The proteins found between 120–180 kDa most likely represent mucins which adhere to the epithelium, as they are similar to the adherent mucins (AMs) from previous reports (Enss et al. 1996). The proteins found between 12 and 70 kDa probably consist of mucin constituents mixed with luminal proteins, the so-called luminal mucins (LMs) (Enss et al. 1996). Smaller molecules (LMs) probably represent the soluble luminal mucus. The AMs form a framework in which smaller molecules might be entrapped.

Luminal mucin might be essential for protection of the intestinal surface, as higher bacterial adhesion to smaller mucus molecules has been observed (Schroers et al. 2008). Mucin degradation by pathogenic bacteria has been suggested earlier for C. carpio (Van der Marel et al. 2008; Schroers et al. 2009) and might largely take place at the carbohydrate side chains of the mucins. In humans, it is known that bacteria with extracellular glycosidases may contribute to the damage of intestinal mucins (Jonas et al. 1977). In clinically healthy C. carpio, bacterial degradation of the protein core of mucus HMGs appears to be limited because secreted and non-secreted mucus have comparable-sized mucins indicated by their similar protein profiles.

However, between different intestinal segments differences in carbohydrate staining could be observed, indicating differences in pH of the mucus. Differences between the first and second segment in the staining intensity of the glycoproteins in the goblet cells support the role of mucus in the defence against pathogens. This fits well in the immunological role, as previously suggested for the second segment (Rombout et al. 1989). The mucus from the intestinal bulb might have also a different function in defence against bacteria as differences in carbohydrate staining to that of first and second intestinal segment could be seen.

A possible different role for mucus in the defence against bacteria is underlined by the different staining for BD2. Staining for BD2 was more pronounced for tissue samples of the first than for the second segment. It is probable that under physiological circumstances, the release of BD2 in the first segment is high enough, so that BD2 molecules can also be functional in the second segment. Staining for BD3 and lysozyme was low and similar between the first and second segment. Therefore, under unchallenged conditions, the humoral substances BD3 and Lys appear to be less important in C. carpio.

Besides the LMs, also AMs were found. Functionally, large molecules (AMs) are regarded to form the mucus layer, which adheres to the epithelium. The main function of these proteins appears to be some kind of stabilizer or carrier for the mucus. In the present study, size areas of the major protein residue between 120 and 180 kD were variable in height, but were a single peak with little variation in retention time. Retention volume of the first peak was similar for samples from the first and second intestinal segment (peak at approximately 150 kDa) but was slightly but significantly larger (approximately 140 kDa) for mucus of the intestinal bulb. Size shifts of around 10 kDa are often caused by a difference in the glycosylation pattern of a protein. The smaller size of the molecules might play a role in food transportation, as smaller molecules are considered to be washed away easier. The intestinal bulb has a food storage function, with the mucus enveloping the food for easier transport. Furthermore, a large part of the digestion has already taken place in the intestinal bulb.

Mucus alterations upon SBM

Soybean meal is used in fish feed, as it is a cheap source of protein. The use of soybean in fish feed, however, is sometimes causing problems, as SBM-containing diets are known to induce an inflammatory response in the hindgut of certain fish species. Contrary to previous observations made with S. salar, C. carpio start to recover or adapt to the SBM feed from the fourth week after the SBM feeding (Uran et al. 2008).

After SBM feeding, the second gut segment of C. carpio did not show external disease symptoms as found previously for carp by Uran et al. (2008). A clear loss of supranuclear vacualistion could not be found. However, a clear increase in goblet cell number in the AB2.5 staining by week three, as found by Uran et al. (2008), could also be observed. This indicates that the carp in this study suffered from an acute but mild enteritis process. In carp, the enteritis process is accompanied by damages to the intestinal epithelium (Uran et al. 2008). In the present study, a higher number of bacteria could be isolated from internal organs after SBM feeding. This might be explained by damage to the intestinal epithelium. The increased bacteria number in the internal organs indicates that the mucosal barrier as first line of defence was compromised, supporting the theory that the intestinal barrier was affected. For humans, increased gut permeability has been described following enteritis. If changes after enteritis persist, a chronic inflammation can develop (Dunlop et al. 2006). Between the control and week 3 after 20 SBM feeding, no difference could be found in bacterial numbers in intestinal organs, indicating that the intestinal barrier function recovers over time.

All samples from the SBM experiment showed a biphasic protein profile with a major protein peak at approximately 150 kDa. The size of the large mucins did not change markedly. However, switching to the 20 SBM diet led to a significant increase in peak height of 150 kDa proteins in non-secreted mucus of the first segment, whereas the amount of 150 kDa in secreted mucus remained relatively stable. The obseved increase in peak height became less over time. Mucus has a high turnover rate, as mucus traps pathogens that can be removed from the body through a constant flow of mucus. The increase in the non-secreted mucus indicates that the SBM diet induces an increased mucus synthesis. The amount of newly synthesized mucus might increase the mucus flow, which would explain why the amount of secreted mucus remains stable. An increased mucus flow stimulates the removal of pathogens from the entire subsequent intestine, which might hereby prevent pathogens from entering the soybean-damaged intestinal epithelium. Changes in mucin levels during enteritis have been described for chronic IBD in humans.

In IBD, genetic mutations in mucin genes, changes in sulphation, degree of glycosylation, mucin mRNA, protein levels and degradation of mucins have been described. Changes of immunological or bacterial factors during an initial or ongoing inflammation can influence mucin production, which could have further adverse effects on mucosal–bacterial interactions, hereby sustaining the chronic character of the inflammation (Einerhand et al. 2002). In ulcerative colitis (a form of IBD), mucin protein levels (Einerhand et al. 2002) as well as the expression of MUC2 were reduced (Van der Sluis et al. 2008). MUC2 is the structural component of the colonic mucus layer in humans (Van der Sluis et al. 2008). Reduced Muc2 levels were also observed in interleukin (IL)-10 knockout mice that develop colitis (Van der Sluis et al. 2008). The expression of mucin 2 proteins and the anti-inflammatory cytokine IL-10 by type-2 T-helper cells and activated macrophages, which induces its expression, seems to be essential in the control of enteritis in mice. The increase in mucus glycoproteins as seen in the first intestinal segment in carp under SBM diet decreased over time, which indicates that mucus composition returns to the state before switching to the SBM diet. An initial up-regulation of IL-10 at week 1, followed by a down-regulation to slightly below or to the level before the enteritis induction was observed previously for C. carpio (Uran et al. 2008).

In the present study, a 20 SBM diet induced changes in the mucus, indicating that the mucosal layer showed altered mucus secretion upon 20 SBM feeding. In the first segment, the amount of sulphated glycoconjugates decreased, suggesting that similar to IBD reports (Einerhand et al. 2002), SBM feeding also leads to changes in mucus sulphation in C. carpio. In addition, mucus from SBM-fed C. carpio showed a different glycosylation pattern as was also reported for IBD (Einerhand et al. 2002). The different glycosylation pattern is indicated by the significantly increased amount of neutral glycoconjugates in the first intestinal segment and the significantly decreased amount of acid and neutral glycoconjugates in the second intestinal segment.

Besides the changes in mucus HMs, also a difference in staining for BD3 could be observed. One week after switching to the SBM diet, the amount of goblet cells that were stained for the antimicrobial peptide BD3 was significantly reduced. This might indicate that BD3 was released to the intestinal lumen to fight pathogens, as BD3 in humans has a strong bactericidal activity (Maisetta et al. 2003). No goblet cells could be stained at week 1 after the diet switch, this might indicate that BD3 was released more rapidly than it could be produced. After the initial depletion of the goblet cells, synthesis and secretion of BD3 seemed to return to their initial equilibrium, as on week 2 or 3, no differences compared with the control could be observed anymore.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

In conclusion, no indication for a different protein core of mucus HMGS for first and second segment could be found. This is in contrast to what is found for humans and other mammalian species analysed. In the present study, only differences in mucus glycosylation could be observed. Along the gut axis, between the intestinal segments and the intestinal bulb, a difference in the size of the major protein peak could be observed. This difference, however, can also be caused by a different glycosylation. A difference between first and second segment could be observed for the antimicrobial peptide BD2.

Furthermore, it was found that SBM diet induces a change in mucins in C. carpio similar to that found in IBD in humans. Initial changes included: changes in mucin composition, the presence of BD3 and of bacteria in internal organs. After the initial changes, most of the values measured returned back to the initial pre-SBM diet values. This might indicate a recovery of the mucosal layer and thus a recovery of the primary barrier between the C. carpio and its surrounding. Changes in the mucosal layer form only a small part in the complex inflammation process of soybean-mediated enteritis with its many symptoms such as abnormal supranuclear vacuolization of the absorptive cells in the intestinal epithelium (Van den Ingh et al. 1991). The recovery of the extrinsic mucus layer may help C. carpio in recovering from SBM enteritis and not developing a chronic inflammation, as through the restoration of the barrier, the C. carpio is no longer constantly exposed to new pathogens.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The authors acknowledge the financial support by the DFG (Deutsche Forschungsgemeinschaft).

References

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
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