Effects of dietary supplementation of chitosan on humoral and cellular immune function in weaned piglets

Animal Feed Science and Technology 186 (2013) 204–208

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Effects of dietary supplementation of chitosan on humoral and cellular immune function in weaned piglets Junliang Li, Binlin Shi ∗ , Sumei Yan, Lu Jin, Yiwei Guo, Yuanqing Xu, Tiyu Li, Xiaoyu Guo College of Animal Science, Inner Mongolia Agricultural University, Huhhot 010018, PR China

a r t i c l e

i n f o

Article history: Received 18 June 2013 Received in revised form 29 September 2013 Accepted 13 October 2013

Keywords: Cellular immunity Chitosan Humoral immunity Weaned Piglets

a b s t r a c t The effects of dietary supplementation of chitosan on humoral and cellular immune function in weaned piglets were investigated. One hundred and eighty piglets weaned at 28 d (Duroc × Large white × Landrace) were assigned randomly to 5 dietary treatments with 6 repetitions in each treatment. The piglets in the 5 treatments were fed on the basal diet supplemented with 0 (control), 100, 500, 1000 and 2000 mg chitosan/kg feed. Results showed that chitosan improved serum immunoglobulin G (IgG) concentrations of piglets in a quadratic dose-dependent manner (P<0.05), and increased serum specific ovalbumin (OVA) IgG contents in a linear or a quadratic dose-dependent manner (P<0.05) on day 28, while serum immunoglobulin A (IgA) and immunoglobulin M (IgM) concentration were not altered. With increasing chitosan, the secretory immunoglobulin A (sIgA) was enhanced in ileum mucosal surfaces in a linear or quadratic manner (P<0.05) on day 14, and was improved quadratically in jejunum mucosal surfaces on day 28 (P<0.05). In addition, chitosan decreased serum concentrations of soluble CD4 (sCD4) in a quadratic dose-dependent manner (P<0.05) and soluble CD8 (sCD8) in a linear or quadratic dose-dependent manner (P<0.05) on day 28. Chitosan quadratically enhanced serum concentrations of interleukin-1 (IL-1) and interleukin-2 (IL-2) on day 14 as well as serum concentrations of tumor necrosis factor-alpha (TNF-␣) on day 28 (P<0.05). These results implied that dietary supplement with chitosan improved humoral and cellular immune responses of weaned piglets in a dose-dependent manner, and in this experiment, the appropriate adding dose of chitosan might be between 500 and 1000 mg/kg. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The immune system of piglet is underdeveloped fully at early age (Heugten et al., 1996), and the ability to resist disease mainly depends on passive immunity from the sow during this time (Rooke and Bland, 2002). Early weaning not only interrupts the supply of immunologically important factors from sow’s milk (Wu et al., 2004), but also impairs the production of antibodies and compromises cellular immune functions (Touchette et al., 2002), which leads piglets to be infected more easily by pathogens. Traditionally, antibiotics were frequently used in the diets of newly weaned pigs for the prophylaxis of infections during the immediate post-weaning period in past decades (Bosi et al., 2011). However, there has been increasing

Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M; IgA, immunoglobulin A; sIgA, secretory immunoglobulin A; sCD4, soluble CD4; sCD8, soluble CD8; IL-1, interleukin-1; IL-2, interleukin-2; TNF-␣, tumor necrosis factor-alpha. ∗ Corresponding author at: College of Animal Science, Inner Mongolia Agricultural University, 306 Zhaowuda Road, Huhhot, Inner Mongolia 010018, PR China. Tel.: +86 471 430 8841; fax: +86 471 431 3717. E-mail addresses: [email protected], [email protected] (B. Shi). 0377-8401/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anifeedsci.2013.10.007

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Table 1 Composition and nutrient levels of the basal diet (air dry basis, %). Ingredients

Content

Nutrients

Level

Corn Soybean meal Wheat Fish meal Corn gluten meal Whey powder Soya bean oil Limestone CaHPO4 NaCl Premixa Total

51.90 16.00 20.00 2.50 2.00 2.00 2.00 0.70 1.00 0.30 1.60 100

Digestible energy (MJ/kg) Crude protein Crude fat Crude fibre Calcium Phosphorus Lysine Methionine + Cystine Threonine

14.32 20.02 3.0 4.2 0.72 0.56 1.35 0.82 0.74

a The premix provides following nutrients per kg diet: Vitamin A, 16,000 IU; Vitamin D3, 2500 IU; Vitamin E, 60 IU; Vitamin K3, 4.5 mg; Vitamin B1, 2.6 mg; Vitamin B2, 8.7 mg; Vitamin B6, 7.0 mg; Vitamin B12, 0.03 mg; vitamin C, 200 mg; Pantothenic acid, 13 mg; Nicotinic acid, 35 mg; Biotin, 0.47 mg; Folic acid, 0.85 mg; Iron, 155 mg; Copper, 35 mg; Zinc, 100 mg; Manganese, 25 mg; Iodine, 0.35 mg; Cobalt, 0.2 mg; Selenium, 0.25 mg; Choline chloride, 750 mg; Phytase, 500 FTU.

pressure on the livestock industry to decrease or discontinue these additions because of the potential development of antibiotic resistance (Davis et al., 2004). Therefore, alternative additives that help develop the immune responses of weaned piglets are highly recommended. Chitosan, a natural and nontoxic alkaline polysaccharide, is formed by the action of chitin deacetylases and is a key structural component of helminths, arthropods and fungi (Synowiecki and Al-Khateeb, 2003). Porporatto et al. (2005) demonstrated that chitosan profoundly affected intestinal mucosal immunity by activating leukocytes. Our previous study found that chitosan improved the humoral and cellular immune functions in broilers (Li, 2009). In piglets, however, there were limited studies evaluating the effect of chitosan on immune function. Therefore, our study was conducted to determine the effect of chitosan on the humoral and cellular immune function of weaned piglets and the appropriate chitosan supplemental level as an immuno-modulating agent. 2. Materials and methods All procedures described in this experiment were approved by Animal Care and Use Committee of Inner Mongolia Agricultural University. 2.1. Experimental design and animal management A total of 180 piglets (Duroc × Large white × Landrace) with an initial average body weight of 7.6 kg were assigned randomly to 5 treatments with 6 repetitions (3 pens of males and 3 pens of females) in each treatment, with 6 piglets in each pen (4.0 m × 4.2 m). The formation of basal diets was showed in Table 1. All diets were offered in meal form. Five dietary treatments supplemented with 0 (control), 100, 500, 1000 or 2000 mg chitosan/kg feed on the basal diet, respectively. Piglets were weaned at the age of 28 d, penned in a temperature-controlled nursery building where temperature was maintained at 26–27 ◦ C and relative humidity was about 65–70%. The weaned piglets had one week of housing and management adaptation before the experimental phase. The experimental period was 28 d. Feed and water were freely available. Chitosan used in this trial was provided by Jinan Haidebei Marine Bioengineering Limited Company (Jinan, China). The deacetylation degree of chitosan was determined to be 85.09%, and the viscosity was 45 cps. 2.2. Sample collection On day 14 and 28, one pig from each replicate of each treatment was randomly selected and blood samples were obtained by puncturing the vena cava. The blood samples were centrifuged at 3000 × g for 20 min at 4 ◦ C to yield serum. Serum was stored at −20 ◦ C until analysis of immunoglobulins, cytokines and sCD4, sCD8. At the beginning of trial, one piglet from each repetition of each treatment was selected randomly and injected with 1 mg ovalbumin/kg BW (Sigma, USA). Blood samples were collected by puncturing the vena cava on day 0 (before injection), 14 and 28 to test the specific OVA antibody concentrations in serum. The blood samples were centrifuged at 3000 × g for 20 min and stored at −20 ◦ C until analysis. For determining the content of sIgA in small intestine mucosa, the pigs used to get blood samples were sacrificed, and the jejunum and ileum were quickly removed, and then the samples were cut and washed with PBS (pH 7.2–7.4). Intestinal mucosa was gently scraped with slide, weighed 1 g and transferred to a centrifuge tube adding 9 mL saline then homogenized by hand. Homogenates were centrifuged at 3000 × g for 20 min, and the supernatant was stored at −20 ◦ C until analysis.

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Table 2 Effects of chitosan on the level of immunoglobulins and OVA-IgG in serum as well as the sIgA of jejunum and ileum mucosal in weaned piglet. Items

Level of chitosan (mg/kg) 0

100

SEM 500

1000

2000

Linear

Quadratic

0.145 0.120

0.491 0.765

0.067 0.024

0.72 0.98

0.069 0.071

0.201 0.862

0.340 0.702

1.38 1.40

1.18 1.39

0.120 0.092

0.971 0.899

0.205 0.540

30.47 196.81 156.37

34.41 211.41 156.57

33.53 161.47 170.29

3.548 15.583 11.942

0.465 0.923 0.016

0.752 0.010 0.035

26.30 23.12

27.63 23.29

27.19 22.56

26.19 21.53

1.192 0.457

0.832 0.383

0.433 0.028

26.92 23.32

27.29 23.90

25.26 23.20

23.62 22.75

0.811 0.671

0.011 0.694

0.012 0.291

IgG(g/L) 14d 28d

7.59 7.88

7.85 8.28

8.32 8.51

8.06 8.31

7.90 8.11

IgM(g/L) 14d 28d

0.76 0.95

0.87 1.03

0.88 1.06

0.77 1.05

IgA(g/L) 14d 28d

1.09 1.31

1.33 1.42

1.35 1.56

31.59 159.41 117.79

29.35 163.73 153.28

sIgA of jejunum (␮g/mL) 14 day 25.22 21.43 28 day sIgA of ileum (␮g/mL) 24.97 14 day 21.12 28 day

OVA-IgG(␮g/L) 0d 14d 28d

P-value

Note: IgG = immunoglobulin G; IgM = immunoglobulin M; IgA = immunoglobulin A; OVA-IgG = ovalbumin IgG; SEM = standard error of the mean.

2.3. Detection of immunoglobulins, cytokines, sCD4 and sCD8 in serum The IgG, IgA and IgM were determined with a porcine ELISA kit (Bethyl Laboratories, Inc., USA) and the minimum detectability was 15.6 mg/L. The IL-1, IL-2 and TNF-␣ were measured with a commercially available porcine ELISA kit from the BioSource International, Inc. (Camarillo, USA). The minimum detectability of IL-1, IL-2 and TNF-␣ were 0.003, 0.01 and 0.03 ng/L, with 12–13%, 7–10% and 11–12% intra- and interassay CV, respectively. The sCD4 and sCD8 were measured with a commercial porcine ELISA kit from the BioSource International, Inc. (Camarillo, USA). The minimum detectable dose was 0.1 U/mL with 9% and 15% intra- and interassay CV, respectively. 2.4. Determination of specific ovalbumin antibody in serum OVA-IgG contents were determined with a porcine ELISA kit from the R & D Systems, Inc. (Minneapolis, USA). Briefly, standard samples were serially diluted and the final concentrations were 120, 80, 40, 20 and 10 ␮g/mL, respectively. Samples were added to testing sample wells. The plate was incubated for 30 min at 37 ◦ C then was washed and added HRP-conjugate reagent. Absorbance at 450 nm after adding stop solution and with in 15 min was read after coloring. 2.5. Specific secretory immunoglobulin A analysis The specific sIgA in small intestine mucosa was determined with a porcine ELISA kit (R&D Systems). Assay procedure was similar with the determination of OVA-IgG. 2.6. Statistical analysis Regression analysis was conducted to evaluate linear and quadratic effects of chitosan on the various response criteria in piglets by using the SAS software 9.0. Trends were considered significant if probability values of P<0.05 were obtained. 3. Results With increasing addition of chitosan, serum IgG concentrations increased in a quadratic dose-dependent manner (P<0.05), while serum IgA and IgM concentrations were not altered (Table 2). Serum OVA-IgG average content of piglets in each group was about 30 ␮g/mL and had no difference between treatments before the trial (Table 2). However, serum OVA-IgG was enhanced by chitosan quadratically (P<0.05) on day 14 and improved linearly or quadratically (P<0.05) on day 28. With increasing chitosan, the sIgA was enhanced in ileum mucosa in a linear or quadratic manner (P<0.05) on day 14, and was improved quadratically in jejunum mucosa on day 28 (P<0.05) (Table 2).

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Table 3 Effects of chitosan on the concentration of sCD4, sCD8 and cytokines in serum of weaned piglets. Items

Level of chitosan (mg/kg)

SEM

0

100

500

1000

2000

sCD4(U/mL) 14d 28d

47.09 47.92

47.02 46.10

43.28 41.61

40.27 41.35

46.47 45.55

sCD8(U/mL) 14d 28d

37.19 42.87

37.62 38.84

39.36 36.85

35.46 36.96

IL-1(ng/mL) 14d 28d

0.28 0.32

0.35 0.36

0.38 0.38

IL-2(ng/mL) 14d 28d

6.59 6.64

6.64 7.01

TNF-␣(ng/mL) 14d 28d

2.09 1.59

2.11 1.72

P-value Linear

Quadratic

2.419 2.390

0.685 0.249

0.107 0.031

41.60 34.62

2.306 1.925

0.242 0.012

0.300 0.028

0.36 0.37

0.30 0.37

0.024 0.025

0.569 0.358

0.028 0.488

7.51 7.13

7.66 7.93

6.64 6.43

0.341 0.509

0.721 0.783

0.036 0.099

2.26 1.91

2.05 2.06

2.03 1.67

0.099 0.142

0.096 0.807

0.142 0.029

The result showed that chitosan decreased serum CD4 in a quadratic dose-dependent manner (P<0.05) and sCD8 in a linear or quadratic manner on day 28 (P<0.05) (Table 3). The maximum decrease of SCD4 in 1000 mg/kg chitosan group and sCD8 in 500 mg/kg chitosan group were 13.71% and 13.79% on day 28, respectively. Chitosan quadratically enhanced serum IL-1and IL-2 on day 14 as well as serum TNF-␣ in piglets on day 28 (P<0.05) (Table 3). In addition, piglets in 500 and 1000 mg/kg chitosan treatments had higher serum sIgA, IL-1, IL-2 and TNF-␣ concentrations compared with other treatments. However, positive effects of chitosan tended to be suppressed when chitosan was increased to 2000 mg/kg.

4. Discussion The humoral immune response is very important for animals, which is effective for protection against most bacterial as well as certain viral infections (McKee et al., 2007). Our results showed that chitosan improved remarkably serum IgG of piglet in a quadratic dose dependent manner. David et al. (2007) indicated that chitosan as an adjuvant enhanced significantly serum IgG titers in mice. Our results were similar to this previous report, and indicated that chitosan improved the production of antibodies by B lymphocytes, which was beneficial for the improvement of humoral immunity in piglets. Serum IgG increased by chitosan was particularly important because it might countervail the decrease of antibodies due to weaning. In our study, the specific antibody response to OVA in chitosan treatments was enhanced significantly on days 14 and 28. Kobayashi et al. (2013) showed that administration of OVA with chitosan microparticles or cationized chitosan induced a high OVA-specific IgA response in mice. This suggested that chitosan could improve animal’s specific immune defense response. We observed that chitosan enhanced the sIgA contents in jejunum and ileum mucosa of pigs. Xu et al. (2004) demonstrated that mice immunized with chitosan-DNA (pcDNA3-VP1) encoding VP1 as an adjuvant produced much higher levels of mucosal sIgA compared to mice treated with pcDNA3-VP1 or pcDNA3. Our results agreed with those of previous study. The sIgA antibodies improved by chitosan might protect efficiently the mucosa of gastrointestinal tract from the pathogens in pigs. Because the mucosal surfaces represent the largest area of exposure of the body to external pathogens, and secretory IgA produced by activated B-cells is the main effector of the mucosal immune system and provides an important first line of defense against most pathogens that invade the body at a mucosal surface (Williams and Gibbons, 1972). The cellular immune response plays an important role in the host response to intracellular pathogens by limiting replication and accelerating clearance of infected cells. The sCD4 and sCD8 in peripheral blood are the soluble form of CD4+ and CD8+ , which are associated with the activation of these cells (Uehara et al., 2003). These soluble forms have been identified as important markers of the activation of T lymphocytes and the occurrence of disease or infection. In the present study, chitosan decreased serum sCD4 and sCD8, which suggested that chitosan could modulate T lymphocytes immune function and maintain health of weaned piglets. Our results demonstrated that the IL-1, IL-2 and TNF-␣ in serum of piglets were enhanced by chitosan. Baek et al. (2007) showed that chitosan increased the production of serum IL-2 and TNF-␣ in elderly adult. Li (2009) indicated that chitosan increased the serum IL-1, IL-2 and TNF-␣ in broilers. In our study, the increasement of serum IL-1, IL-2 and TNF-␣ implied chitosan enhanced cell-mediated immunity in piglets. In addition, our previous study indicated that chitosan could quadratically improve growth in weaned pigs (Xu et al., 2013), which may be a reflection of immune function improved by chitosan. However, further experiments need to be done to verify the effects of chitosan on immune functions in weaned piglets.

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5. Conclusions It is concluded that chitosan probably improved the humoral and cellular immune function in weaned piglets, and the appropriate adding dose of chitosan might be between 500 and 1000 mg/kg. Conflict of interest Author disclosures: Junliang Li, Binlin Shi, Sumei Yan, Lu Jin, Yiwei Guo, Yuanqing Xu and Tiyu Li have no conflicts of interest. Acknowledgments The authors gratefully acknowledge the support of the National Natural Science Foundation of China (Project No. 31060310). References Baek, K.S., Won, E.K., Choung, S.Y., 2007. Effects of chitosan on serum cytokine levels in elderly subjects. Arch. Pharm. Res. 12, 1550–1557. Bosi, P., Merialdi, G., Scandurra, S., Messori, S., Bardasi, L., Nisi, I., Russo, D., Casini, L., Trevisi, P., 2011. Feed supplemented with 3 different antibiotics improved food intake and decreased the activation of the humoral immune response in healthy weaned pigs but had differing effects on intestinal microbiota. J. Anim. Sci. 89, 4043–4053. David, A.Z., Connie, J.R., Kenneth, W.H., Jeffrey, S., John, W.G., 2007. Chitosan solution enhances both humoral and cell-mediated immune responses to subcutaneous vaccination. Vaccine 11, 2085–2094. Davis, M.E., Maxwell, C.V., Erf, G.F., Brown, D.C., Wistuba, T.J., 2004. Dietary supplementation with phosphorylated mannans improves growth response and modulates immune function of weanling pigs. J. Anim. Sci. 82, 1882–1891. Heugten, E., Coffey, M., Spears, J.W., 1996. Effects of immune challenge, dietary energy density, and source of energy on performance and immunity in weanling pigs. J. Anim. Sci. 74, 2431–2440. Kobayashi, T., Fukushima, K., Sannan, T., Saito, N., Takiguchi, Y., Sato, Y., Hasegawa, H., Ishikawa, K., 2013. Evaluation of the effectiveness and safety of chitosan derivatives as adjuvants for intranasal vaccines. Viral Immunol. 26, 133–142. Li, H.Y., 2009. Effects of Chitosan on Immune Function in Broilers Chickens and The Underlying Mechanisms. Ph.D. Thesis. Inner Mongolia Agricultural University, Huhhot. McKee, A.S., Munks, M.W., Marrack, P., 2007. How do adjuvants work? Important considerations for new generation adjuvants. Immunity 27, 687–690. Porporatto, C., Bianco, I.D., Silvia, G.C., 2005. Local and systemic activity of the polysaccharide – chitosan at lymphoid tissues after oral administration. J. Leukoc. Biol. 78, 62–69. Rooke, J.A., Bland, I.M., 2002. The acquisition of passive immunity in the new-born piglet. Livest. Prod. Sci. 78, 13–23. Synowiecki, J., Al-Khateeb, N.A., 2003. Production, properties, and some new applications of chitin and its derivatives. Crit. Rev. Food Sci. Nutr. 43, 145–171. Touchette, K.J., Carroll, J.A., Allee, G.L., Matteri, R.L., Dyer, C.J., Beausang, L.A., Zannelli, M.E., 2002. Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: I. Effects on the immune axis of weaned pigs. J. Anim. Sci. 80, 494–501. Uehara, S., Gothoh, K., Handa, H., Tomita, H., Tomita, Y., 2003. Immune function in patients with acute pancreatitis. J. Gastroenterol. Hepatol. 18, 363–370. Williams, R.C., Gibbons, R.J., 1972. Inhibition of bacterial adherence by secretory immunoglobulin A: a mechanism of antigen disposal. Science 177, 697–699. Wu, G., Knabe, D.A., Kim, S.W., 2004. Arginine nutrition in neonatal pigs. J. Nutr. 134, 2783-2790. Xu, W., Shen, Y., Jiang, Z.G., Wang, Y., Chu, Y.W., Xiong, S.D., 2004. Intranasal delivery of chitosan-DNA vaccine generates mucosal sIgA and anti-CVB3 protection. Vaccine 22, 3603–3612. Xu, Y.Q., Shi, B.L., Yan, S.M., Li, T.Y., Guo, Y.W., Li, J.L., 2013. Effects of chitosan on body weight gain, growth hormone and intestinal morphology in weaned pigs, Asian- Australas. J. Anim. Sci. 26, 1484–1489.