Effects of antibacterial peptide on humoral immunity in weaned piglets

Abstract

The aim of this study was to investigate the effects of dietary antimicrobial peptide (AMP) supplementation on the serum IgG, IgM, IgA, classical swine fever antibody (CSF-Ab), and total complement (CH50) levels in weaned piglets. Ninety weaned piglets were randomly allotted to five groups for 28-day study. Blood samples were collected at 32, 39, 46, and 53 days of age. The results showed that AMP as dietary additive increased the levels of IgG, IgM, IgA, CSF-Ab, CH50 among the whole experimental period, and the effects of AMP supplementation were dose-dependent. The study showed that supplementing weaned piglets diets with AMP enhanced the humoral immune responses of weaned piglets by improving the levels of classical swine fever virus antibody, CH50, and immunoglobulins. We suggest the dosage of AMP was 500 mg/kg.

Keywords:

• antibacterial peptide
• classical swine fever antibody
• total complement
• immunoglobulin
• weaned piglets

1. Introduction

Previously, piglets are weaned occurs over several weeks or months. However, in the modern pig industry, piglets are weaned early between 3 and 4 weeks of age to prevent sow-originated infectious diseases and maximize the whole herd production (Dong & Pluske, 2007; Kim, McPherson, & Wu, 2004). This practice presents a tremendous challenge to neonatal piglets, whose immune functions of the weaned piglets always matured at 7 weeks of age (Yang and Schultz, 1986). During this critical period, some viral diseases, such as classical swine fever and porcine respiratory and reproductive syndrome, could cause greater morbidity and mortality in weaned piglets, resulting in significant economic loss. Antibiotics were frequently used for the prophylaxis of infections in past decades (Bosi et al., 2011). However, there has been increasing pressure on the livestock industry to decrease or discontinue these additions because of the potential development of antibiotic resistance (Davis, Maxwell, Erf, Brown, & Wistuba, 2004). Therefore, how to improve the immunity of weaned piglets is an important problem.

Antimicrobial peptide (AMP) is a series of short chain peptides composed of dozens of amino acid residues. It shows a broad range of activity against gram-negative and gram-positive bacteria, fungi, mycobacterium (Zasloff, 2002), virus (Huang, Kingsolver, Avadhanula, & Hardy, 2013), tumor (Yan et al., 2012), parasite (Torrent, Pulido, Rivas, & Andreu, 2012), and immune function enhancing (Yu et al., 2010). In some studies, it has been reported that dietary supplementation of different AMP to chicken, rabbit, piglet diets improved the levels of immunoglobulins (Liu, Yang, Hua, Liu, & Wang, 2012; Lv, Yuan, Cai, & Yin, 2011; Wu et al., 2012) and alexin C₃ (Guo, Yang, et al., 2012).

On the basis of the foregoing, we hypothesized that dietary supplementation with AMP enhances the humoral immune function in weaned piglets. This hypothesis was tested by determining the serum IgG, IgM, IgA, classical swine fever antibody, and CH50 levels after initiation of the treatment.

2. Materials and methods

2.1. Materials

The AMP used in the present study was provided by Rota Bioengineering Co., Ltd., Sichuan, China. The antibacterial peptide (ABP) was composed of swine defensin (DHYICAKKGGTCNFSPCPLFNRIEGTCYSGKAKCCIR) and a fly ABP (ATCDLLSGTGVKHSACAAHCLLRGNRGGYCNGRAICVCRN) at a blending ratio is 50%. Astragalus polysaccharide (AP; net content, 65%) was purchased from Centre Biology Co., Ltd., Beijing, China. All chemicals used were of the greatest purity grade available.

2.2. Animals and experimental design

Ninety weanling piglets (Landrace × Yorkshire × Duroc; 21 ± 2 days of age) were purchased from Xin Qiao Agricultural Science and Technology Development Co., Ltd., Chengdu, Sichuan, China, and inoculated intramuscularly at the age of 21 days with classical swine fever live vaccine (C-strain, rabbit origin, Guangzhou Winsun Pharmaceutical Co., Ltd, China). All piglets (45 males and 45 females) were acclimatized for 5 days prior to usage. Weanling piglets (average body weight, 8.24 ± 0.67 kg) were randomly allotted to five treatment groups and fed a standard diet (Table 1) (NRC, 1998). Each treatment group consisted of three replicates with six piglets (three males and three females) per replicate, and each replicate was housed in an individually elevated pens with wire flooring. Dietary treatments were as follows: (1) control; (2) control + 400 mg/kg AP; (3) control + 250 mg/kg AMP; (4) control + 500 mg/kg AMP; (5) control + 1000 mg/kg AMP. The temperature (26–27°C) and relative humidity (65–70%) were kept constant. Food and water were provided ad libitum during the acclimatization period and throughout all of the study proper, and the experimental lasted for 28 days. Two piglets (one male and one female) from each replicate were selected randomly to take blood samples for analysis at 32, 39, 46, and 53 days of age. All piglets used in this experiment were approved to be healthy. All experimental manipulations were undertaken in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals.

Table 1. Composition of experimental diets^a.

Ingredient	(%)
Corn	53.15
Soybean meal (44% crude protein)	25.40
Whey powder	6.00
Fish meal (62.5% crude protein)	4.50
Soybean oil	2.80
Spray-dried porcine plasma (78% crude protein)	3.00
Dicalcium phosphate	2.0
Limestone	1.40
Salt	0.25
L-Lysine HCl	0.15
DL-Methionine	0.35
Premix^b	1.00

^a Calculated nutrient composition (as-fed basis): 3280 kcal of DE/kg, 1.14% lysine, and 0.32% methionine. Analyzed nutrient composition (as-fed basis): 21.1% crude protein, 0.68% Ca, and 0.59% P.

^b Provided the following per kilogram of complete diet: vitamin A, 2250 IU; vitamin D₃, 1050 IU; vitamin E, 16 IU; vitamin K₃, 0.9 mg; vitamin B₁₂, 0.03 mg; riboflavin, 4.5 mg; niacin, 30 mg; pantothenic acid, 25 mg; choline chloride, 400 mg; folic acid, 0.30 mg; thiamin, 2 mg; pyridoxine, 7 mg; biotin, 0.20 mg; Zn, 80 mg; Mn, 22 mg; Fe, 80 mg; Cu, 225 mg; I, 0.50 mg; and Se, 0.25 mg.

2.3. Measurement of classical swine fever antibody (CSFV-Ab), CH50, and immunoglobulins

At 32, 39, 46, and 53 days of age, blood samples from six piglets of each treatment group (two piglets per replicate) were collected between 8.00 am and 10.00 am for the determination of serum parameters. 2 mL blood samples were collected from the anterior vena cava of piglets, and allowed to clot at 37°C for half an hour and centrifugalized at 1000 × g at 4°C for 20 min, then the serum was collected and stored in Eppendorf tubes at –20°C. Total CSFV-Ab, CH50, and immunoglobulins were determined in serum, using enzyme-linked immunosorbent assay (ELISA) kits purchased from Wuhan ColorfulGene Biological Technology Co., Ltd (Wuhan, China). All operations were in accordance with the instructions of the kits.

2.4. Statistical analysis

Results are reported as means ± standard deviation (M ± SD). Statistical analysis was performed by one-way analysis of variance using SPSS 19.0 software. Duncan's multiple range test was used to compare differences among the treatment groups, and P < 0.05 was considered statistically significant.

3. Results

During the entire experiment period, the levels of CSFV-Ab of the treatment groups were higher than that of the control group (Table 2), and the values of CSFV-Ab of the AMP groups were higher than that of the AP group (except the level of CSFV-Ab of AMP group I was lower than that of the AP group on day 32). The level of CSFV-Ab of the AMP group II was markedly higher (P < 0.05) than that of the control group at 39, 53 days of age. The level of CSFV-Ab of the AMP group III was significantly higher (P < 0.05) than that of the control group among the whole experiment period, and superior (P < 0.05) to that of the AP group at 32, 53 days of age. Moreover, the levels of CSFV-Ab of the AMP linearly improved as the level of dietary ABP increased.

Table 2. The effects of ABP on the classical swine fever antibody levels in piglets (optical density ratio) (means ± SD).

	Treatments
Time	Control group	AP group	AMP group I	AMP group II	AMP group III
32 days	0.25 ± 0.02^c	0.28 ± 0.02^b	0.27 ± 0.03^ab	0.31 ± 0.04^ab	0.33 ± 0.04^a
39 days	0.31 ± 0.03^b	0.34 ± 0.02^ab	0.35 ± 0.03^ab	0.36 ± 0.01^a	0.37 ± 0.03^a
46 days	0.34 ± 0.04^b	0.36 ± 0.05^ab	0.38 ± 0.047^ab	0.41 ± 0.05^ab	0.44 ± 0.07^a
53 days	0.30 ± 0.07^c	0.38 ± 0.03^bc	0.39 ± 0.03^abc	0.42 ± 0.04^ab	0.46 ± 0.04^a

Note: Different lowercase letters represent P < 0.05.

During the whole experiment period, the levels of CH50 of the treatment groups were significantly higher (P < 0.05) than that of the control group (Table 3), and the values of CH50 of the AMP groups were markedly higher (P < 0.05) than that of the AP group (except the level of CH50 of the AMP group I on day 32, 39, and the level of CH50 of the AMP group II on day 32 were lower than that of the AP group). Moreover, the values of CH50 of the AMP linearly improved as the level of dietary ABP increased.

Table 3. The effects of ABP on the CH50 levels in piglets (U/mL) (means ± SD).

	Treatments
Time	Control group	AP group	AMP group I	AMP group II	AMP group III
32 days	345.91 ± 10.61^c	435.65 ± 15.86^a	410.34 ± 14.13^b	434.44 ± 14.22^a	445.40 ± 17.48^a
39 days	361.03 ± 22.06^c	459.98 ± 16.60^b	453.43 ± 10.80^b	485.78 ± 17.20^a	503.23 ± 11.32^a
46 days	370.52 ± 14.50^e	487.02 ± 4.38^d	508.70 ± 7.34^c	547.33 ± 11.17^b	571.53 ± 10.16^a
53 days	380.30 ± 20.82^d	503.06 ± 8.55^c	572.47 ± 11.74^b	605.58 ± 21.00^a	612.43 ± 7.48^a

Note: Different lowercase letters represent P < 0.05.

At 39, 46, and 53 days of age, the levels of IgG of the AMP group II, the AMP group III, and the AP group were significantly higher (P < 0.05) than those of the control group and the AMP group I (Table 4). And there were no differences (P > 0.05) between the value of IgG of the AMP group I and that of the control group on days 32, 39, and 53. The levels of IgG of the AMP group II and the AMP group III were higher than that of the AP group among the whole experiment period, but there were no differences (P > 0.05). Moreover, the values of IgG of the AMP linearly improved as the level of dietary ABP increased.

Table 4. The effects of ABP on the immunoglobulin levels in piglets (g/L) (means ± SD).

		Treatments
Items	Time	Control group	AP group	AMP group I	AMP group II	AMP group III
	32 days	3.44 ± 0.14^a	3.49 ± 0.19^a	3.46 ± 0.11^a	3.58 ± 0.54^a	3.66 ± 0.23^a
	39 days	3.63 ± 0.28^b	4.37 ± 0.41^a	3.69 ± 0.14^b	4.50 ± 0.32^a	4.77 ± 0.46^a
IgG
	46 days	3.69 ± 0.33^c	4.90 ± 0.33^a	3.95 ± 0.21^b	4.94 ± 0.42^a	5.20 ± 0.35^a
	53 days	3.47 ± 0.59^b	4.79 ± 0.35^a	3.74 ± 0.53^b	4.89 ± 0.42^a	5.13 ± 0.32^a
	32 days	0.71 ± 0.02^c	0.79 ± 0.02^c	0.78 ± 0.03^c	1.01 ± 0.08^b	1.13 ± 0.09^a
	39 days	0.76 ± 0.04^d	0.93 ± 0.05^c	0.90 ± 0.05^c	1.15 ± 0.08^b	1.27 ± 0.09^a
IgM
	46 days	0.90 ± 0.06^c	1.11 ± 0.05^b	1.00 ± 0.04^c	1.19 ± 0.07^a	1.28 ± 0.010^a
	53 days	0.88 ± 0.04^d	1.10 ± 0.06^b	0.99 ± 0.05^c	1.18 ± 0.06^b	1.25 ± 0.06^a
	32 days	0.52 ± 0.02^a	0.53 ± 0.02^a	0.52 ± 0.01^a	0.53 ± 0.02^a	0.54 ± 0.05^a
	39 days	0.53 ± 0.09^b	0.59 ± 0.08^ab	0.58 ± 0.07^b	0.64 ± 0.04^a	0.68 ± 0.04^a
IgA
	46 days	0.50 ± 0.02^c	0.65 ± 0.04^b	0.64 ± 0.03^b	0.70 ± 0.02^b	0.76 ± 0.05^a
	53 days	0.45 ± 0.04^b	0.67 ± 0.06^a	0.52 ± 0.03^b	0.68 ± 0.04^a	0.73 ± 0.04^a

Note: Different lowercase letters represent P < 0.05.

Except there were no differences (P > 0.05) between the value of IgM of the AMP group I on day 32 and 46, the value of IgM of the AP group on day 32 and that of the control group (Table 4), the values of the treatment groups were significantly higher (P < 0.05) than that of the control group among the whole experiment period. The level of IgM of the AMP group I was markedly lower (P < 0.05) than that of AP group at 39, 53 days of age. The values of IgM of the AMP group II and the AMP group III were significantly higher (P < 0.05) than that of the AP group among the whole experiment period (except the value of IgM of the AMP group II on day 53). Moreover, the values of IgM of the AMP linearly improved as the level of dietary ABP increased.

The levels of IgA of the treatment groups were higher than that of the control group among the whole experiment period (except the value of IgA of the AMP group I on day 32; Table 4). At 46, 53 days of age, the value of IgA of the AP group was markedly higher (P < 0.05) than that of the control group. At 39, 46, and 53 days of age, the levels of IgA of the AMP groups were significantly higher (P < 0.05) than that of the control group (except the level of IgA of the AMP group I on day 39, 53). Moreover, the values of IgA of the AMP linearly improved as the level of dietary ABP increased.

4. Discussion

The level of antibody reflects the humoral immunity of organism (Waters, Terrell, & Jones, 1986). Antibody usually appears after vaccination not only in the blood but also locally about 1 week. In our study, supplementation with AMP and AP could increase the levels of CSFV-Ab, although the effects of CSFV-Ab of middle dosage (500 mg/kg) and high dosage (1000 mg/kg) of AMP were better than that of AP. This result was consistent with Zhang's report on foot and mouth disease virus antibody, where 6.25–50 mg/kg AP were administered to mice (Zhang et al., 2010). In line with the results of the present study, Jiang Yang, Qiu, Zhang, and Dai (2013) reported that oral administration of 160 mg/kg AMP cecropins to piglets could significantly increased (P < 0.05) the blocking rate, positive rate, and qualified rate of CSFV antibody. Similarly, artificial AMPs also could induce high levels of antigen-specific antibodies after the co-injection of AMPs with a commercially available influenza vaccine in mice (Fritz et al., 2004). Increased (P < 0.05) the serum antibody titers of Newcastle disease virus (NDV) and Avian influenza virus (AIV) were also reported in chickens injecting physiological saline supplemented with 0.1–0.5 mg AMP of rabbits sacculus rotundus (Wang et al., 2007). Moreover, Yang et al. (2006) reported significantly enhanced (P < 0.05) in the antibody response to infectious bursal disease virus vaccine (IBDV) in chickens receiving drinking water supplemented with chicken intestinal AMPs (1 mg/mL) 21 days following IBDV vaccine administration.

Complement system plays an important role in specific immune and nonspecific immune of organism. CH50 can reflects the humoral immune function of body (Janeway, Travers, Walport, & Shlomchik, 1999). To the best of our knowledge, no reports on the effects of ABP on CH50 of weanling piglets are yet available. In our study, piglets fed with AMP and AP markedly improved the values of CH50, although the effects of CH50 of middle dosage (500 mg/kg) and high dosage (1000 mg/kg) of AMP were better than that of AP. Our results suggested that AMP could enhance humoral immune responses of weaned piglets by improving complement system. Previous research indicates that weaned piglets fed 1000 and 1500 mg/kg Achyranthes bidentata polysaccharide show enhanced serum contents of C₃, C₄ (Chen, Liu, & He, 2009). Similarly, New Zealand rabbits fed diets supplemented with 200 mg/kg AMP were reported to have greater (P < 0.05) the complement 3 (C₃) contents (Guo, Yang, et al., 2012). Moreover, Shan, Wang, Wang, Liu, and Xu (2007) reported that oral administration of 1000 mg/kg AMP (lactoferrin) to weaned piglets could significantly enhanced (P < 0.05) in the complement 4 (C₄) contents.

The serum immunoglobulin titer is an important indicator of humoral immunity (Kong et al., 2007). The results of our study showed that AMP and AP increased the levels of IgG, IgM, and IgA. While, the effects of IgG, IgM, IgA of middle dosage (500 mg/kg) and high dosage (1000 mg/kg) of AMP were more obvious, and were better than that of AP. This result is consistent with Guo's report on serum contents of immunoglobulin M (IgM) and immunoglobulin G (IgG), where 60 mg/kg AP was administered to immunosuppressive chickens (Guo, Liu, et al., 2012). In line with the results of the present study, Shan et al. (2007) reported that oral administration of 1000 mg/kg AMP (lactoferrin) to weanling piglets could markedly increased (P < 0.05) the levels of IgG, IgM, and IgA. Similarly, weanling piglets fed diets supplemented with 10 mg/kg AMP were reported to have greater (P < 0.05) concentrations of serum IgG (Wang, Wu, Feng, 2011). Increased (P < 0.05) the contents of IgG and IgM in serum were also reported in chickens receiving drinking water supplemented with intestinal AMPs (1 mg/mL) from days 4–10 and days 10–17, respectively (Yang et al., 2006). Moreover, Lv et al.'s (2011) positive effect of dietary supplementation of 200, 400, and 600 mg/kg AMP on concentrations of serum IgM and IgA in hisex hens. In contrast to the results of the present study, Yoon et al. (2014) reported that no significant differences on serum IgG, IgA, and IgM concentrations in weanling piglets fed with 60 mg/kg AMP-A3 and P5, respectively. This discrepancy results may be attributed to variations in the type of AMP used, the level of dietary supplementation, and the mode of action of AMP.

The levels of CSFV-Ab, CH50, and immunoglobulins of 1000 mg/kg AMP group were higher than those of 500 mg/kg AMP group, but there were no differences (P > 0.05). And considering the price of AMP. Therefore, we suggest the dosage of AMP was 500 mg/kg.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The present work was supported by the Changjiang Scholars & Innovative Research Team of Ministry of Education of China Funds [grant number IRTO848].