Active or passive immunisation against cholecystokinin-33 stimulates growth of pigs

Abstract

The purpose of this study was to determine the effect of active or passive immunisation against cholecystokinin (CCK) peptide on feed intake and body weight gain in pigs. For active immunisation, recombinant CCK-33 concatenate polypeptide was employed to immunise pigs, while passive immunisation was achieved by egg powders containing CCK-specific antibodies from laying hens that were immunised with recombinant CCK-33 peptide. Enzyme-linked immunosorbent assay showed that the active immunised pigs had significantly higher levels of CCK antibodies in serum than the control ones (p<0.05). Actively or passively immunised pigs had significantly higher body weight, average daily gain and average daily feed intake than that of the control ones (p<0.05). The results suggest that active or passive immunisation against CCK-33 peptide has stimulatory effects on growth of pigs. Thus, immunisation against CCK-33 may be used as an alternative way to enhance pig growth.

Keywords:

• CCK
• appetite
• feed intake
• growth
• immunisation
• pig

Introduction

A growing body of evidence suggests that signals from the gastrointestinal tract is directly involved in the control of feed intake of animals (Ritter, 2004a; Strader & Woods, 2005). The gastrointestinal signals are collectively called ‘satiety signal’, including dozens of gastrointestinal hormones that are known to be secreted from the endocrine cells lining the gastrointestinal tract in response to the physicochemical properties of ingested feed passing along the lumen (Gomez et al., 1996; le Roux & Bloom, 2005; Liddle, 1997; Lloyd, 1994; McIntosh, 1985; Srivastava, Kamath, Barry, & Dayal, 2004; Woods, 2004). The gastrointestinal hormones may signal the central nervous system in either endocrine or paracrine manner (Harrold, 2004). Subsequently, the central nervous system interprets these signals and sends signals to numerous organs in the periphery to modulate visceral functions and feeding behaviour (Figlewicz, Lacour, Sipols, Porte, & Woods, 1987; Moran, Ladenheim, & Schwartz, 2001).

Cholecystokinin (CCK) is one of the gastrointestinal hormones, secreted primarily in two forms, CCK-33 and CCK-8 by the I-cells within the duodenal and jejunal mucosa (Polak et al., 1975). Previous studies showed that nutrient-stimulated release of CCK by enteroendocrine cells triggers several downstream events, including pancreatic enzyme secretion, excretion of bile into the intestine, slowing of gastric emptying and inhibition of feed intake (Liddle, 1997; Matson & Ritter, 1999; Ritter, Covasa, & Matson, 1999). Additionally, CCK was shown to function as a trophic hormone in supporting the growth of stomach, pancreas and intestine (Barrowman, 1975; Enochs & Johnson, 1977; Kuntz, Pinget, & Damge, 2004). Vagus nerves appear to be necessary for CCK-mediated inhibition of feed intake, since destruction of these neuron fibres attenuates the reduction of feed intake by CCK. It has been suggested that CCK may act on its target tissues via two G-protein coupled receptors, CCK-A and CCK-B, both of which are expressed by the vagus nerves (Ritter, 2004a, b; Sun & Ferguson, 1997).

Neutralisation of plasma CCK (active immunisation with CCK-8-hsG conjugate) or blockage of CCK receptors was shown to stimulate animal feed intake and daily body weight gain in several animal species including growing swine (25.6–77 kg, Yorkshine and Large white) and gilts (Pekas, 1991; Pekas & Trout, 1990, 1993), but decrease feed intake in lambs (Spencer, 1992). In the present study, we examined whether active or passive immunisation against plasma CCK affects feed intake and daily body weight gain of growing pigs (43–80 kg, Du×(chang×da)).

Materials and methods

DNA constructs

The open reading frame of chicken CCK-33 was amplified by PCR using the specific primers pair CCKup (5′-CAT GGA TCC GGT TCT ACT GGC CGC T-3′)/CCKdn (5′-CAT GAG CTC CCA AAA TCC ATC CAG CC-3′; the homology of chicken CCK-33 to pig CCK-33 was as high as 95%). The PCR product was digested with Bam HI and Sac I and cloned into plasmid pRSET-A (Invitrogen, Karlsruhe, Germany) to generate recombinant plasmid pRSET-1CCK. The insert of pRSET-1CCK was then excised out by two double restriction digestions of Bam HI/Hind III and Bgl II/Hind III and joined together into pRSET to generate pRSET-2CCK. Following a similar procedure, pRSET-4CCK was generated. The final product pRSET-4CCK was verified by PCR, enzyme digestive identification and sequencing.

Expression and purification of concatenated CCK-33 peptide

To express concatenate CCK peptide, the plasmid pRSET-4CCK was transformed into BL-21 Escherichia coli cells. A single ampicillin-positive clone was picked and grown in LB broth. When OD₆₀₀ reached approximately 0.5, IPTG was added to the bacterial culture to induce expression of recombinant CCK protein. Samples of the bacterial culture were removed at 2, 3 and 4 hours after addition of IPTG, lysed and analysed by Western blot.

IPTG-induced bacterial cells were collected by centrifugation, resuspended in 40 ml/l buffer B (8 M urea, 0.1 M NaP, pH 8.0), and lysed by sonication. After centrifugation for 20 min at 10,000 rpm in a 50 ml Oakridge tube, the supernatant was collected and added to a column loaded with 50% Ni-NTA resin. The column was washed with 25–30 ml buffer B and 40 ml of buffer C (8 M urea, 0.1 M NaP, pH 6.3) to remove bacterial proteins. The bound protein was eluted with buffer F (8 M urea, 5% glacial acetic acid) and analysed by SDS-PAGE and Western blotting.

Preparation of oil-emulsion vaccine

The vaccine was prepared with purified concatenated CCK-33 peptide of 0.5 mg/ml in oil-emulsion adjuvant.

Preparation of the CCK-33-specific antibody

Sixty-four healthy laying hens were housed in the experimental area of the Guangdong Wen's Food Co., Ltd. (Guangdong, China) and allotted randomly to two groups, Group 1 was used for control and Group 2 for immunisation with recombinant CCK-33 peptide. Each group consisted of eight replicates with four hens in each replicate. The hens in Group 2 received the vaccine at 1 ml/bird. Control hens were injected at the same ages with physiological saline prepared in oil emulsion. The hens were inoculated at 20 d intervals for three times. For detection of immune responses, blood samples and eggs were collected from the immunised and control birds at 28, 48, 68 and 94 testing days and analysed by enzyme-linked immunosorbent assay (ELISA). Eggs containing CCK-33-specific antibody were collected for preparation of egg powder.

Immunisation of pigs actively by vaccine and passively by CCK-33-specific antibody

A total of 96 triple hybrid pigs of 75 d were housed in the experimental area of the Guangdong Wen's Food Co., Ltd. and allotted randomly to four groups and each treatment consisted of three replicates with eight pigs in each replicate. Their diets and nutrition level were listed in Table 1. Pre-trial feeding was done for 7 d, and then the pigs were immunised at 82 d. For active immunisation, each pig was injected intraperitoneally with 2 ml of the oil-emulsion vaccine at 82 and 110 d of age, respectively. Control pigs were injected at the same ages with physiological saline prepared in oil-emulsion adjuvant. Blood samples were collected from the immunised and control animals at 128 d and analysed by ELISA. For passive immunisation, egg powder containing CCK-33-specific antibody was added to the feed of pigs at 100 g/t, and pigs of another groups were fed egg powder at 100 g/t without CCK-33-specific antibody. All the experiments were ended at 128 d.

Table 1. Basal diet and nutrition level of pigs.

Diet ingredients	82–110 d	111–128 d
Corn meal (%)	67.9	66.16
Soybean meal (%)	23	23.8
Wheat bran (%)	–	2
Rap meal (%)	5	4
Lime powder (%)	0.7	0.7
Phosphate powder (%)	1.4	1.34
Pre-mix (%)	2	2
Nutrition level	–	–
Crude protein (%)	17	17.5
Digestive energy (KJ/kg)	3120	3140
Lysine (%)	0.9	0.95
Methionine + cystine (%)	0.56	0.55
Threonine (%)	0.62	0.63
Calcium (%)	0.7	0.7
Available phosphorus (%)	0.38	0.38

Measurements of average daily gain, average daily feed intake and feed to gain ratio

At the beginning and termination of the experiment each pig was weighted at 9 am after food was withheld for one night. The amount of feed consumed was recorded during the experiment to determine average daily gain, average daily feed intake and feed to gain ratio.

Statistical analyses

Statistical analyses were performed using one-way ANOVA procedure of SPSS 11.0 software. Differences between individual means were determined by Duncan's new multiple range test. Probability value (p) < 0.05 was considered significant. Data are expressed as the mean±SE.

Results

Verification of pRSET-4CCK

Figure 1 shows that the recombinant plasmid of pRSET-1CCK, pRSET-2CCK and pRSET-4CCK was digested with Bam HI and Sac I and yielded about 120, 230 and 450 bp bands in size, respectively, which were 1CCK, 2CCK and 4CCK. The recombinant pRSET-4CCK was sequenced (Shanghai Boya Ltd.) and conformed to our original design essentially (data not shown).

Figure 1.  Identification of pRSET-1CCK, pRSET-2CCK and pRSET-4CCK. M, DL2000 marker; 1, Bam HI/Sac I-digested pRSET-1CCK, 1CCK was about 120 bp in size; 2, Bam HI/Sac I-digested pRSET-2CCK, 2CCK was about 230 bp in size; 3, Bam HI/Sac I-digested pRSET-4CCK, 4CCK was about 450 bp in size.

Expression of 4CCK protein

SDS-PAGE analysis of the 4CCK fusing protein was displayed in Figure 2. As expected, the expression of CCK protein was induced by 1 mmol/L IPTG and its molecular was about 20 kDa. Further testing of the expressed protein with Western blot using rabbit anti-CCK hyper-immune serum (Sigma, USA) revealed a specific protein band of about 20 kDa in size (Figure 3), which demonstrated that the fusion protein could react with CCK-33-specific antibody.

Figure 2.  SDS-PAGE analysis of 4CCK expression in different time. M, protein molecular weight; 1–5, expression products of E. coli containing recombinant plasmids induced with 0, 1, 2, 3 and 4 h. The molecular weight of 4CCK was about 20 kDa.

Figure 3.  Western blot analysis of 4CCK expressed products. M, pre-stained protein standard; 1, 4CCK expressed products.

Detection of anti-CCK antibody in hen serum and yolk

Tables 2 and 3 show the serum and yolk antibody levels against CCK-33. It can be seen that the CCK-specific antibody in serum and yolk in treated group was much higher than that of control except that anti-CCK antibody in yolk at 94 d was relatively low. As CCK was an endogenous protein, it is plausible that a longer period of immunisation raised tolerance that made anti-CCK antibody in yolk at 94 d was relatively low.

Table 2. Detection of hens anti-CCK antibody level in serum (n=32).

	Testing days
Groups	28	48	68	94
Control group	1.009±0.018^a	1.059±0.041^a	0.993±0.035^a	1.038±0.023^a
Immunised group	1.918±0.029^b,C	2.622±0.052^b,B	3.087±0.060^b,A	2.073±0.037^b,C
^a,bMeans in the same column are significantly different (p<0.05). ^A,B,CMeans in the same row are significantly different (p<0.05). Note: Data are presented as mean±SE.

Table 3. Detection of hens anti-CCK antibody level in yolk (n=32).

	Testing days
Groups	28	48	68	94
Control group	0.950±0.015^a	0.988±0.077^a	0.961±0.065^a	1.007±0.043^a
Immunised group	1.874±0.036^b,C	2.751±0.018^b,B	3.126±0.051^b,A	1.472±0.046^b,D
^a,bMeans in the same column are significantly different (p<0.05). ^A,B,CMeans in the same row are significantly different (p<0.05). Note: Data are presented as mean±SE.

Detection of anti-CCK antibody in pig serum

The immune responses of pigs were determined by ELISA (Table 4). At 128 d, significant levels of antibody against CCK were detected in the plasma of pigs immunised with recombinant CCK-33 peptide. The results indicated that pigs immunised with 0.5 mg/ml of concatenated recombinant CCK-33 peptide produced significantly high levels of anti-CCK antibody in their blood stream.

Table 4. Comparison of antibody levels between control and immunised groups at 128 d (n=24).

Groups	Antiserum levels
Control	1.018±0.02^a
Immunised group	2.063±0.09^b
^a,bMeans are significantly different (p<0.05). Note: Data are presented as mean±SE.

Analysis of the body weight and feed intake of pigs

We compared the body weight, average daily gain, average daily feed intake and feed to gain ratio of the actively or passively immunised pigs to that of the control animals (Table 5). It shows that the final weight, average daily gain and average daily feed intake of the actively or passively immunised pigs were significantly higher than that of the control pigs (p<0.05). There was no difference between the two control groups (p>0.05), and there were also no difference between the actively immunised pigs and passively immunised pigs (p>0.05). There was no significant difference in feed to gain ratio among the four groups (p>0.05).

Table 5. Comparison of body weights, feed intakes and feed efficiencies between control and treated groups (n=24).

Experimental groups	Initial weight (kg)	Final weight (kg)	Average daily gain (kg/d)	Average daily feed intake (kg/d)	Feed to gain ratio
Control	43.050±0.177	79.220±0.225^a	0.770±0.004^a	2.060±0.026^a	2.675±0.011
Active immunised group	43.256±0.141	81.533±0.372^b	0.815±0.002^b	2.168±0.028^b	2.660±0.029
Fed egg powder without CCK antibody	42.790±0.222	79.448±0.201^a	0.779±0.013^a	2.078±0.059^a	2.667±0.054
Passive immunised group	42.783±0.167	81.878±0.112^a	0.832±0.003^b	2.160±0.004^b	2.592±0.005
^a,bMeans in the same column are significantly different (p<0.05). Note: Data are presented as mean±SE.

Discussion

CCK is an endogenous protein and anti-CCK antibody was produced in the serum of hens. According to the results of hen antibody against CCK, a longer period of immunisation raised the negative feedback of CCK antibody reaction with endogenous CCK, which stimulated animals to produce more endogenous CCK, thus it may caused the concentration of CCK antibody relatively low. In addition, CCK and gastrin are highly similar peptides, CCK antibodies may also bind and neutralise gastrin hormones (Colucci et al., 2005; Stubbs et al., 2002). While gastrin stimulates gastric acid release and is not known to directly control food intake, it is possible that anti-gastrin activity could influence acidity and thereby influence overall intake and weight gain, which indicated that alternative mechanisms for CCK antibodies increased feeding weight gain may exist.

Feed intake in animals is thought to be controlled by a complex network of endocrine signals. Stimulation of feed intake has been explored to maximise the growth potential of animals. Our findings suggest that neutralisation of endogenous CCK-33 hormone through active immunisation increases feed intake and stimulates body growth in pigs, which is in line with the evidence generated in growing swine (26.5–77 kg), barrows and gilts (Pekas, 1991; Pekas & Trout, 1990, 1993), and supports the hypothesis that endogenous CCK functions as a negative appetite regulator (Ritter et al., 1999). Given that no apparent pathological condition was observed in the immunised animals, we propose that immunisation against plasma CCK may be used as a means to stimulate feed intake and growth in pigs.

We found that passive immunisation against CCK-33 for improving feed intake and growth of pigs were equally effective to active immunisation but was relatively more expensive. Passive immunisation has potential advantages over vaccination in that higher doses of antibody can be administered in feed, its apparent pathological effect such as the stress and ache on animal was less and the antibody dose can be controlled so that individual variability in serum antibody levels might be minimised, and the onset of effect is immediate. In addition, the usefulness of egg yolk immunoglobulin Y (IgY) has been assessed for therapeutic application by passive immunisation through oral ingestion of IgY because of its broad stability towards heat, acid and pepsin (Shin et al., 2002). Although the principle and procedure of IgY absorption in gastrointernal tract is still unknown, the phenomenon has been observed and utilised in prevention or control of many infections (Kuroki et al., 1997; Marquardt et al., 1999; Sunwoo, Nakano, Dixon, & Sim, 1996). For these reasons, passive immunisation with egg yolk-derived antibodies is of interest as a possible therapeutic alternative to vaccination. Our reports suggest that passive immunisation with CCK antibodies is a feasible strategy for improvement of feed intake and growth of pigs.

Acknowledgements

This study was supported by grant from Natural Science Group Foundation (04205804) and Science Technology Strategic Plan (2006A20403002) of Guangdong, People's Republic of China.