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Direct Enhancement of the Phagocytic and Bactericidal Capability of Abdominal Macrophage of Chicks by β-1,3–1,6-Glucan

K.-L. Chen^*, B.-C. Weng, M.-T. Chang^*, Y.-H. Liao^*, T.-T. Chen^* and C. Chu^,1

^* Department of Animal Science, and Department of Microbiology and Immunology, National Chiayi University, 300 University Road, Chiayi 60004, Taiwan

¹ Corresponding author: cschu{at}mail.ncyu.edu.tw

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Salmonella enterica serovar Enteritidis is the major zoonotic and intracellular pathogen. Different strategies have been developed to prevent the S. Enteritidis infection. The β-1,3–1,6-glucan of Schizophyllum commune was used as an immunological booster to determine the minimal dietary level of β-glucan that would restrict S. Enteritidis infection through the effects of β-glucan on the activity of macrophages and direct physical protection of the intestine. One-day-old male Single Comb White Leghorn chicks were used in all trials. In trials 1 and 2, the 0.1% β-1,3–1,6-glucan treatment completely eliminated the viable S. Enteritidis from spleen and liver in an oral challenge of 10⁸ S. Enteritidis without any harmful effect on BW, serum proteins, and immunoglobulin. Instead of a 21-d feeding period of β-glucan, a 14-d treatment was enough to eliminate the S. Enteritidis in spleen and liver. In trial 3, an increase in the relative weight of bursa of Fabricius and phytohemagglutinin-P-inducing cutaneous basophil hypersensitivity was observed (P < 0.05). In trials 2, 3, and 4, the direct or indirect effect of β-1,3–1,6-glucan on abdominal macrophages was examined. Sterilized 3% Sephadex G-50 was injected to induce abdominal (peritoneal) phagocytes in chicks fed with or without 0.1% β-1,3–1,6-glucan. Significantly increased phagocytic and bactericidal capability to S. Enteritidis was found in abdominal macrophages either pretreated or in vitro treated with 0.1% β-1,3–1,6-glucan. In conclusion, in addition to the physical properties to block S. Enteritidis entrance, 0.1% dietary β-1,3–1,6-glucan may enhance the host defense to S. Enteritidis by directly upregulating the phagocytosis and bactericidal activity of abdominal macrophages in chicks.

Key Words: chick • β-glucan • macrophage • phagocytosis • Salmonella

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Salmonella enterica serovar Typhimurium and Enteritidis are the broad-host-range pathogens that cause salmonellosis in humans and animals. The consumption of Salmonella-contaminated eggs and poultry meats results in endemic food-borne disease in humans. Among serovars isolated from chicken, S. Enteritidis is the predominant serovar to cause such disease (De Buck et al., 2004; Hernandez et al., 2005), which can also be isolated from other contaminated foods such as those found in a bakery (Lu et al., 2004). Recently, great efforts have been applied to reduce S. Enteritidis and S. Gallinarum infection (Chacana and Terzolo, 2006). Through boosting immune system, natural β-glucans differing in linkage of glucose polymers in (1,3), (1,4), or (1,3) (1,6) direction and in side chain structure (Cheung et al., 2002) of yeast, fungus, grains, and seaweed can enhance host defense to microbial infection including bacteria (Lowry et al., 2005), virus (Jung et al., 2004), protozoan (Yun et al., 2003), and fungus (Kruse and Cole, 1992).

The β-glucan-inducing immune responses in poultry differed depending on β-glucan types. For example, β-glucan of Saccharomyces cerevisiae elevates phagocytic activity and lymphocyte proliferation (Guo et al., 2003) and increases the CD8 and TCR 1 cells respectively of chicks (Chae et al., 2006). Another rich polysaccharide of mushroom, lentinan, promotes splenocyte proliferation and interleukin-2 production in broilers (Chen et al., 2003). In addition, β-1,3-glucan of Schizophyllum commune significantly increases the chemotaxis activity of macrophages (Cheng et al., 2004) and protects chicks from S. Enteritidis infection by enhancing the capabilities of heterophiles in peripheral blood (Lowry et al., 2005). Further, abdominal macrophage system plays another important role in defense of pathogens entering through intestinal epithelial cells. However, the mechanism by which β-1,3-glucan of Schizophyllum commune reduces the S. Enteritidis infection via abdominal macrophages still remains unclear. Therefore, the purposes of this study were (1) to determine the minimal dietary level of β-glucan that would restrict S. Enteritidis infection, (2) to determine the possible protective effects of β-glucan in chicken through an elevated immunity or a direct physical protection in intestine, and (3) to investigate the protective and persistent immuno-stimulatory effects of β-glucan on macrophage. Dietary supplementation of 0.1% β-1,3–1,6-glucan was enough not only to eliminate the S. Enteritidis from liver and spleen in vivo, but also to enhance the prolonged phagocytic and bactericidal capabilities of abdominal (peritoneal) macrophages.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Feeding of Birds

One-day-old male Single Comb White Leghorn chicks were used and reared in wire floored battery brooders with electrical heat. The diets formulated to meet NRC (1994) requirements (CP 18.1%, 2,900 kcal of ME/kg) and water were provided ad libitum. The β-1,3–1,6-glucan of Schizophyllum commune was purchased from Taito Co. (Tokyo, Japan) with a purity of 52.2%.

Trial 1. Protection Effect of the β-1,3–1,6-Glucan on S. Enteritidis Infection

Experimental Design. One hundred twenty chicks were randomly assigned into 4 groups treated, respectively, with 0, 0.025, 0.05, and 0.1% dietary β-glucan. Each treatment contained 3 replicates and each replicate included 10 chicks. Body weight and the relative weight of lymphoid organs were measured for all chicks, which were further used for the S. Enteritidis challenge test.

S. Enteritidis Challenge Test. The S. Enteritidis (OU7586) with 60-kb Salmonella virulence plasmid has a low intraperitoneal LD₅₀ (<10² bacteria) in mouse virulence test and was found to be highly infectious to chicks in a preliminary experiment with liver and spleen pathology. Therefore, OU7586 was chosen for the challenge test. On d 14, one milliliter of 10⁸ bacteria of OU7586 was gavaged into each chick. The next day, 3 chicks of each treatment were killed for 7 continuous days. Dietary β-glucan was supplemented for 21 d. The number of viable Salmonella in liver and spleen was measured to determine the protective effect of β-glucan. To avoid the sampling error and standardize the sampling, 0.1 g of left lobe liver and the spleen was taken and pulverized with 0.5 mL of sterilized saline. Then 100 µL of the aliquot was plated on MacConkey agar (Difco, BD, Sparks, MD). After being incubated at 37°C for 18 h, white bacterial colonies were counted and further characterized by Salmonella serogroup D O-antigen (Difco, BD) to ensure they were Salmonella positive.

Trial 2. Short-Term Effect of 0.1% β-Glucan on S. Enteritidis Infection and Phagocytic Assay Experimental Design

To verify the reduction of S. Enteritidis invasion by dietary β-glucan through cell-mediated immune response or physical blockade of bacterial invasion, 132 chicks were randomly divided into 3 groups as follows: 1) control group: chicks fed the diet without β-glucan from 1 to 21 d; 2) BG14d group; chicks fed with 0.1% β-glucan from d 1 to 14 only; and 3) BG21d group: chicks fed with 0.1% β-glucan throughout the 21-d experimental period. There were 4 replications in each group.

S. Enteritidis Challenge Test

On d 14, 36 birds of each dietary treatment were subjected to bacterial challenge test as described above. Four chicks were then killed each day for 7 continuous days. The BW, the relative weight of lymphoid organs, and viable Salmonella in spleen and liver were measured.

Analysis of Phagocytic and Bactericidal Capability

Collection of Abdominal (or Peritoneal) Phagocytic Cells. To verify the effect of dietary β-glucan on the cell-mediated immune response, abdominal (or peritoneal) phagocytic cells (macrophage) were used in an ex vivo phagocytic assay. From d 22, two chicks of the remaining 8 unchallenged chicks were killed each day for 4 continuous days to collect abdominal phagocytic cells (mainly macrophage). Following the methods of Konjufca et al. (2004) to enrich abdominal phagocytic cells, a dose of sterilized 3% Sephadex G-50 was infused (1 mL/100 g of BW) into the peritoneal cavity 1 d before euthanization. Chicks were killed by cervical dislocation. After disinfection of abdominal area with 75% alcohol, the abdominal skin and muscle lining were removed open, and then 3 mL of a 0.5 U/mL heparin saline solution (Sigma-Aldrich Co. Taiwan) was injected to flush the peritoneal area. The flushing solutions from 2 chicks were mixed and centrifuged at 400 x g and 4°C for 10 min to collect the phagocytic cells. Then cell density was adjusted to reach the concentration of 10⁶ cells/mL by hemocytometer with fresh RPMI-1640 medium and 10% fetal bovine serum (Sigma-Aldrich Co. Taiwan).

Ex Vivo Evaluation of the Phagocytic Effect. One milliliter of 10⁶ macrophages was seeded in each well of 24-well plates and cultured overnight at 37°C and 5% CO₂. Then 107 bacteria of OU7586 was added into each well, and the mixture was centrifuged at 450 x g and 37°C for 5 min to increase the attachment of OU7586 onto macrophages. The plates were incubated for 1 h at 37°C and 5% CO₂. Following the method of Chang and Ou (2002) to eliminate the extracellular bacteria, cells were washed with fresh RPMI-1640 medium twice and finally fresh RPMI-1640 medium containing 1 mg/mL of gentamycin (Sigma-Aldrich Co. Taiwan) was added and allowed to react for 1 h. After washing with PBS, intracellular Salmonellae were released from phagocytic cells by treating with 1% sodium deoxycholate immediately [reaction time (t) = 0] or after different culture periods (t = 3 and t = 23). One hundred microliters of supernatant was plated on the MacConkey agar, and viable S. Enteritidis were counted after incubation at 37°C for 18 h. The colony-forming units were recorded and analyzed.

Trial 3. Analysis of Immunological Protein and Reaction as Well as Ex Vivo Phagocytic Assay Experimental Design

To study the other immunological effects and repeat the ex vivo phagocytic assay, 40 chicks were fed with 0 or 0.1% β-glucan. On d 21, twelve chicks of each treatment were used to evaluate the change in serum protein fractions, immunoglobulins, and the cutaneous basophil hypersensitivity (CBH). Starting at d 22, two chicks of each treatment were killed each day for 4 d, providing 8 chicks per treatment. Phagocytic and bactericidal capabilities of abdominal macrophages were evaluated by the methods described in trial 2.

Measurement of Serum Protein, Immunoglobulin, and Cutaneous Basophil Hypersensitivity. Peripheral blood was collected via wing vein. Serum proteins were measured by Titan Gel Serum Protein Kit and gel electrophoresis (Helena Laboratories, Beaumont, TX). The ratio of serum proteins was determined by a Helena Titian Gel photo scanner at wavelength = 595 nm. Following, the level of IgA and IgM of the same serum was determined by ELISA kit (Bethyl Co., Montgomery, TX). To avoid interference between these 2 assays, one week recovery period was allowed. Furthermore, type IV hypersensitivity (CBH) was performed by subcutaneous injection of 100 µL sterilized phytohemagglutinin-P (PHA-P, Sigma-Aldrich Co., Taiwan) containing 100 µg of PHA-P into the toe web between the second and third digit. The same volume of sterilized saline was injected into the same location of the other leg as a negative control. The increment of skin thickness was measured and analyzed according to the method of Corrier and DeLoach (1990).

Trial 4. In Vitro Effects of β-Glucan on Abdominal Macrophages

The effects of β-glucan on abdominal macrophages were studied by using 16 β-glucan-free 22-d-old chicks. Abdominal macrophages of 4 chicks were collected and mixed for 4 continuous days, and then the capabilities of phagocytic and bactericidal effects were determined as described in trial 2. However, abdominal macrophages were cocultured with or without 0.1% β-glucan for 24 h to stimulate the immune response.

Statistical Analysis

Treatment differences were calculated by using Duncan’s new multiple range test to compare the means among treatments according to the method of Steel and Torrie (1960).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The Effects of β-Glucan on BW (Trials 1 and 2)

Due to the consistency of 1,3–1,4 β-glucan of grains, it can decrease feed efficiency through interacting with digesta (Yun et al., 1997; Józefiak et al., 2004). By contrast, 1,3–1,6 β-glucan (up to dietary 0.1% β-glucan) of Schizophyllum commune did not show any deleterious effect on BW gain between chicks treated with or without β-glucan in trial 1 and 2, although chicks were challenged with S. Enteritidis (Table 1). Similar results were reported in chicks receiving levels of 0.0125% to 0.1% β-glucans of Schizophyllum commune, which did not have any detrimental growth performance to those individuals receiving no β-glucan supplementation (Cheng et al., 2004; Chen et al., 2006). These data indicate that 0.1% glucans did not have any harmful effect due to growth performance in the analysis. Therefore, the effect of glucans on S. Enteritidis invasion was studied.

Continuous Supplementation of β-Glucan Before Challenge (Trials 1 and 2). After oral challenge, viable S. Enteriditis was consistently isolated from liver and spleen in control group and bacterial count was higher in the spleen (Tables 2 and 3). In contrast, continuously feeding of β-1,3–1,6-glucan of Schizophyllum commune resulted in a significant reduction and even disappearance of viable S. Enteritidis with increasing level of β-glucan level (Tables 2 and 3). At the β-glucan level of 0.05%, the incidence of S. Enteritidis was seen in the spleen only the first 2 d postchallenge and was absent in the liver. Finally, 0.1% β-glucan was enough to prevent Salmonella invasion in the liver and spleen.

Effect of Dietary β-Glucan on Lymphoid Organs (Trials 1 and 2), Serum Protein, Immunoglobulins, and Cutaneous Basophil Hypersensitivity (Trial 3)

Bursa of Fabricius and thymus are 2 important lymphoid organs. The bursa of Fabricius is the site of B-cell maturation and differentiation responsible for production of immunoglobulin and humoral immune response and the thymus is the site for T-cell maturation (Glick, 2000; Tizard, 2000). Early reports revealed that fungal β-glucans of Schizophyllum commune and beer yeast can increase the relative weight of thymus and the bursa of Fabricius (Guo et al., 2003). In the 0.1%-β-glucan group fed for 21 d, there was a significantly (P < 0.05) increase in the relative weight of the bursa of Fabricius in trial 1, and when fed for 14 d in trial 2 (Table 1). However, 0.1%-β-glucan treatment only increased numerically in the relative weight of thymus in trials 1 and 2. These results suggest that 0.1%-β-glucan might enhance the B-cell and T-cell maturation. There was no significant change in serum proteins and immunoglobulin (Table 4). In contrast, β-glucans of other sources such as oats increase the concentration of serum IgG, IgG₁, IgG₂, IgM, and IgA (Yun et al., 2003). The PHA-P-inducing CBH was significantly increased in chicks treated with 0.1% β-glucan after the 48-h treatment, but not the 24-h treatment (P < 0.05; Table 4). A similar increase was found in chicks fed with β-glucan at 72 h postintradermal injection of PHA-P (Guo et al., 2003), suggesting that dietary β-glucan can increase cell-mediated immunity in vivo.

A. Pretreatment with β-Glucan (Trials 2 and 3). Macrophage functions include phagocytic capability as determined by measuring intracellular bacterial number per macrophage at the beginning and bactericidal effect determined by measuring the number of viable intracellular bacteria over different experimental time courses. The 3% Sephadex G-50-inducing abdominal macrophages and Salmonella-engulfed macrophages were shown in Figure 1. The macrophages of both β-glucan-pretreated groups significantly increased 34 to 37% of phagocytic capability (P < 0.05) in trial 2 (Table 3); however, only minor increase of phagocytosis after treatment of S. Enteritidis 1 h later (t = 0) in trial 3 (Figure 2A). Better phagocytic capacity to Salmonella may be coincident with the upregulated bactericidal effect of β-glucan-treated macrophages against intracellular pathogens. Our results showed a significant decrease of the surviving phagocytosed S. Enteritidis in the β-glucan-treated group (P < 0.05; 8.0 x 10⁴ cfu for 0.1% β-glucan-treated chicks vs. 1.5 x 10⁵ cfu for the control group; Figure 2A). A similar increase of phagocytic ability up to 17 to 23% and bactericidal killing was observed in β-glucan-treated heterophils, a major phagocytic leucocyte in blood, to S. Enteritidis (Guo et al., 2003; Lowry et al., 2005). However, viable phagocytosed S. Enteritidis was only mildly decreased at t = 23 (Figure 2a). This time-course related bactericidal effect may indicate that β-glucan-pretreated macrophages presented the primed and enhanced bactericidal functions to kill bacteria early (t = 3); however, such bactericidal effect could not be maintained persistently (t = 23). Next, whether or not β-glucan directly or indirectly induces phagocytic and bactericidal effects on abdominal macrophages was examined.

View larger version (53K): [in this window] [in a new window]   Figure 1. Morphology of abdominal macrophages after in vitro 24-h culture (A) and abdominal macrophage with phagocytosed Salmonella Enteritidis at reaction time (t) = 3 (B). Cells were stained by Liu stain with 1,000x amplification.

View larger version (15K): [in this window] [in a new window]   Figure 2. Phagocytic and bactericidal capability of abdominal macrophages in vivo pretreated (A, trial 3) or in vitro treated (B; trial 4) with 0.1% β-glucan. Abdominal (peritoneal) macrophages were collected from 2 chicks daily, and this experiment was performed repeatedly for 4 sequential days. Similar macrophage results were obtained for each day, and values represent the means ± SD (n = 8). *Indicates significant difference between control and β-glucan-treated groups at same time period, P < 0.05 (trials 3 and 4). t = reaction time.

In conclusion, dietary supplementation of 0.1% β-1,3–1,6-glucan of Schizophyllum commune enhanced the chicks’ host defense to S. Enteritidis invasion through a macrophage-modulating mechanism and possibly physical protection of β-glucan in the intestine. Upregulated phagocytic and bactericidal capabilities of abdominal macrophages to S. Enteritidis may be through direct interaction between β-1,3–1,6-glucan and macrophages.

	ACKNOWLEDGMENTS

 
We thank the National Science Council of Taiwan for financially supporting this project. This project number is NSC 92-2313-B-415-022.

Received for publication April 7, 2008. Accepted for publication June 1, 2008.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chen, K.-L. Articles by Chu, C. Search for Related Content PubMed PubMed Citation Articles by Chen, K.-L. Articles by Chu, C.