- Email: [email protected]
Active immunisation of lambs with a monoclonal antibody against clenbuterol
Livestock Production Science 68 (2001) 87–91 www.elsevier.com / locate / livprodsci
Short communication
Active immunisation of lambs with a monoclonal antibody against clenbuterol A.F. Carson a
a,b ,
*, C.T. Elliott a,c , D.P. Mackie a,c , W.J. McCaughey a,c
Department of Agriculture and Rural Development for Northern Ireland, Hillsborough, Co. Down BT26 6 DR, Northern Ireland, UK b Agricultural Research Institute of Northern Ireland, Hillsborough, Co. Down BT26 6 DR, Northern Ireland, UK c Veterinary Sciences Division, Stoney Road, Belfast, BT3 4 SD, Northern Ireland, UK Received 14 August 1998; received in revised form 7 June 1999; accepted 28 April 2000
Abstract An experiment was undertaken with 50 Texel 3 Suffolk-Cheviot lambs (5468.8 days of age) to investigate the effects of active immunisation with a murine monoclonal antibody against clenbuterol on growth and carcass characteristics. Animals on treatments 1 and 2 each received 0.1 mg of clenbuterol antibody while animals on treatments 3 and 4 received 0.1 mg of antibody encapsulated within a synthetic polymer. Diethylaminoethyl (DEAE)-dextran was used as the adjuvant in treatments 1 and 3 and saponin in treatments 2 and 4. Control animals were immunised with saponin only. Four immunisations were given at 4-week intervals. Animals were slaughtered 4 weeks after the final immunisation. Each vaccine evoked a similar level of antibody response while the control group showed no titres. Lamb growth rate did not vary significantly between the vaccinated and control groups. Dressing proportion was higher (P , 0.05) in treatments 1 and 4 compared with the control group. Carcass length was greater (P , 0.001) in each of the vaccinated groups compared with the control group. None of the carcass fat depth measurements varied significantly between the control and vaccinated groups. 2001 Elsevier Science B.V. All rights reserved. Keywords: Clenbuterol; Anti-idiotype; Lambs; Carcass
1. Introduction Immunisation procedures can be used to generate antibody responses against a wide variety of antigens, including antibodies. Studies have shown that a proportion of anti-antibodies can react with the antigen combining site (idiotype) of the original
*Corresponding author. Tel.: 1 44-28-9268-2484; fax: 1 4428-9268-9594.
antibody and can therefore sometimes mimic (as an internal image) the action of the original antigen (Jurn, 1974). The objectives of the present study were firstly to determine if immunisation with a murine monoclonal antibody against the beta-agonist clenbuterol generated an anti-idiotype response in lambs, secondly to investigate the relative effectiveness of selected adjuvant formulations in generating an antibody response and thirdly to investigate the effects of anti-idiotype responses on growth and carcass characteristics in lambs.
0301-6226 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 00 )00206-2
88
A.F. Carson et al. / Livestock Production Science 68 (2001) 87 – 91
2. Materials and methods
2.1. Vaccine formulation and treatments A panel of monoclonal antibodies to clenbuterol was produced by the immunisation of a BALB / c mouse with a clenbuterol-transferrin conjugate. After five immunisations (at 3-week intervals) the mouse was killed and the spleen removed and fused with a mouse myeloma by the method of Teh et al. (1984). One of the clones (8B11) secreting clenbuterol antibody was selected (based on titre and cross reactivity profiles) for the production of ascites fluid for use in the present study. For each dose of treatment 1 vaccine, 2 ml of diethylaminoethyl (DEAE)-dextran (Sigma, Poole, UK) (2% solution) was added dropwise, while stirring, to 2 ml of clenbuterol antibody (0.05 mg / ml) at room temperature. For each dose of treatment 2 vaccine, 2 ml of saponin (‘Quil A’: Superfos, Vaalsgard, Denmark) (1 mg / ml) was added dropwise, while stirring, to 2 ml of clenbuterol antibody (0.05 mg / ml) at room temperature. For treatment 3 vaccine (encapsulated antigen plus DEAE-dextran), nanosphere preparation was achieved by emulsion polymerisation (Rodgers et al., 1998). For each vaccine dose, encapsulated antibody (0.1 mg) was diluted in saline (0.05 mg / ml) and to this solution was added 2 ml of DEAE-dextran (2% solution). For each dose of treatment 4 vaccine (encapsulated antigen plus saponin), 2 ml of saponin (1 mg / ml) was added dropwise, while stirring, to 2 ml of clenbuterol antibody (0.05 mg / ml) at room temperature. Control treatment animals received 2 ml of saponin (1 mg / ml).
2.2. Animals and treatments Fifty Texel 3 Suffolk-Cheviot lambs (n 5 19 rams, n 5 31 ewes) with an initial age of 5468.8 days and weight of 20.664.99 kg were allocated to five treatment groups. These groups were balanced for gender, age and live weight. Lambs were grazed with their mothers as a single group on perennial ryegrass-based swards. At approximately 90 days of age, all lambs were weaned and grazed together as a single group.
Four immunisations, administered by intramuscular injection, were given at 4-week intervals. Jugular blood samples (10 ml) were collected immediately prior to the first immunisation and at 14 days after each of the immunisations. The heparinised plasma was assayed for antibodies against the anti-clenbuterol antibody by means of a dissociation enhanced lanthanide fluorimmunoassay (DELFIA). Animals were weighed at 14-day intervals throughout the study and 4 weeks after the fourth immunisation animals were sent for slaughter. Carcasses were commercially graded for conformation (EUROP classification) and fat class (1 5 low fat cover; 5 5 high fat cover). Dressing proportion and linear carcass measurements were recorded as detailed by Carson et al. (1999).
2.3. Clenbuterol antibody assay A purified clenbuterol–human serum albumin conjugate was prepared as described by Elliott et al. (1994). This conjugate was labelled with europium (Elliott et al., 1994) and the resultant complex (CBLHSA-Eu 31 ) was stored in an equal volume of glycerol at 2 208C until required. Microtitre plates (Wallac, Turku, Finland) were coated with 100 ml of mouse anti-rabbit IgG (Sigma) diluted in 10 ml 50 mM carbonate / bicarbonate buffer pH 9.5. Plates were incubated at 378C for 2 h in a microtitre plate incubator / shaker. After a single wash with a 0.9% saline, 1% Tween 20 solution, excess reagent was tapped out onto clean tissue paper. In triplicate, a range of clenbuterol standards (0, 10, 25, 50, 125 and 1250 pg / well) prepared in distilled water were added to microtitre plate wells (25 ml / well). All other wells received 25 ml of distilled water. Into all wells was pipetted 50 ml of an anti-clenbuterol polyclonal antiserum raised in a rabbit, diluted to 1 / 20 000 in a 50 mM Tris–HCl buffer pH 7.4 containing 5 mg / ml BSA (assay buffer). All wells then received 50 ml of CBl-HSAEu 31 diluted 1 / 600 in assay buffer. To the wells of standards was then added 25 ml of new born calf serum (Gibco Ltd., Paisley, Scotland). Plasma samples taken from experimental animals were then added to the remaining wells (25 ml / well in triplicate). Plates were shaken gently at room tempera-
A.F. Carson et al. / Livestock Production Science 68 (2001) 87 – 91
ture for 2 h after which they were washed six times in wash solution. Into each well of the microtitre plates was added 100 ml of europium enhancement solution (Wallac). After a 10-min shake, plates were transferred into a time resolved fluorometer (Wallac 1234 reader) and the fluorescent intensity in each well measured at 630 nm. Standards were used to construct a spline fit standard curve and concentrations of anti-idiotype present in each sample were calculated and quantified in terms of clenbuterol equivalents (ng equiv).
2.4. Statistical analyses
89
Lamb growth rate (calculated by regression) did not vary significantly between the vaccinated and control groups (Table 2). However, dressing proportion was affected by immunisation. Dressing proportion was higher (P , 0.05) in treatments 1 and 4 than in the control group. Carcass length was greater (P , 0.001) in each of the vaccine treatments compared with the control group. None of the carcass fat parameters varied significantly between the control and vaccine treatments.
4. Discussion
The results were analysed by analysis of variance (Genstat, 1993) corresponding to the 5 (treatments) 3 2 (gender) factorial design of the experiment. Initial weight of the lambs was used as a covariate. Antibody responses at each time interval were analysed separately. As there were no significant treatment 3 gender interactions, only main effect means are presented in the tables.
3. Results There were no significant antibody responses generated by the vaccines after the first immunisation (Table 1). However, after the second immunisation, antibody responses (P , 0.05) were recorded in treatments 2 and 3 (vs. controls). After a fourth immunisation, antibody responses (P , 0.01) were recorded in all vaccine treatments.
In the present study, anti-idiotype antibodies to clenbuterol had no effect on lamb growth rate. The effects of beta-agonists on growth rate have been found to be variable. In some studies a significant increase in liveweight gain has been reported (Moloney et al., 1995), while in others, no effect has been observed (Koohmaraie et al., 1996). Moloney et al. (1995) suggested that the response to betaagonists may depend on the growth potential of the animals. The lamb genotype used in the present study may be classified as having a high growth potential and thus have a reduced capacity to respond to the anabolic effects of beta-agonists. Dressing proportion was increased by beta-agonist administration in some studies (Moloney et al., 1995). In the present study, the increase (42–49 g / kg) in dressing proportion in treatments 1 and 4 compared with the controls is in the order of magnitude reported by Warriss et al. (1989) (40
Table 1 Antibody responses to immunisation (ng equiv) Time interval b
PrePost Post Post Post a
first second third fourth
Control
ND 0.0 0.0 A 0.0 A 0.0 A
Treatment groups a
Gender
Significance of effects
1
2
3
4
S.E.D.
Males
Females
S.E.D.
Treatment
Gender
ND 0.1 0.5 AB 0.4 AB 0.8 B
ND 0.1 0.7 B 0.6 B 0.8 B
ND 0.0 0.7 B 0.4 AB 0.8 B
ND 0.0 0.5 AB 0.6 B 0.9 B
– 0.04 0.26 0.23 0.28
ND 0.1 0.4 0.4 0.5
ND 0.0 0.6 0.4 0.8
– 0.02 0.17 0.17 0.18
– NS ** * ***
– NS NS NS NS
1, antibody plus DEAE-dextran; 2, antibody plus saponin; 3, encapsulated antibody plus DEAE-dextran; 4, encapsulated antibody plus saponin. b Blood samples taken immediately prior to first immunisation and 14 days after each immunisation. A,B Treatment group means with the same superscript within the same row are not different (P . 0.05). ND, not detectable.
A.F. Carson et al. / Livestock Production Science 68 (2001) 87 – 91
90
Table 2 Carcass characteristics in the treatment groups Control
Treatment groups a
Gender
Significance of effects
1
2
3
4
S.E.D.
Males
Females
S.E.D.
Treatment
Gender
Growth rate (g/day) Dressing proportion (g/kg) Carcass weight (kg) Conformation classification b Fat classification c Kidney 1 Channel fat (g) Kidney weight (g) Kidney weight (g/kg half carcass weight)
224 488 A 22.1 3.4 4.3 382 141 3.1
241 530 B 23.7 3.5 3.6 505 128 2.7
218 523 AB 23.7 3.5 4.0 577 129 2.4
211 496 AB 23.2 3.5 4.2 383 129 3.2
218 537 B 23.2 3.5 4.0 466 137 2.4
15.3 20.9 1.20 0.43 0.33 95.9 17.0 0.49
241 504 24.0 3.4 4.2 450 130 2.5
205 525 22.4 3.5 3.9 475 136 3.1
9.6 13.1 0.75 0.27 0.21 60.0 10.7 0.31
NS * NS NS NS NS NS NS
*** * * NS NS NS NS *
Carcass linear measurements (mm) Carcass length Width of barrel Width of shoulder Leg length Circumference of buttocks
519 A 246 220 284 462
570 B 251 222 283 484
569 B 248 226 275 487
548 B 251 218 268 491
559 B 251 223 271 485
13.9 10.5 8.3 7.8 15.1
556 252 224 276 486
550 247 219 276 478
8.7 6.6 5.2 4.9 9.4
*** NS NS NS NS
NS NS NS NS NS
3.4 2.4
3.5 2.9
3.9 3.9
2.7 2.7
3.1 3.5
1.10 0.70
3.2 3.2
3.4 3.0
0.69 0.44
NS NS
NS NS
6.1 6.8 6.5 12.9
6.6 7.7 7.3 13.7
7.8 8.2 7.7 13.6
6.1 6.8 6.3 11.3
7.4 8.2 8.4 12.9
1.43 1.38 1.25 1.77
7.3 7.2 7.0 12.0
6.3 7.9 7.6 13.8
0.90 0.86 0.78 1.11
NS NS NS NS
NS NS NS NS
Linear fat measurements (mm) Fat depth over Longissimus dorsi 35 mm from midline 50 mm from midline Fat depth at loin (mm) 35 mm from midline Above Gluteus medius At Obliqus internus abdominis Tissue depth (mm) a
1, antibody plus DEAE-dextran; 2, antibody plus saponin; 3, encapsulated antibody plus DEAE-dextran; 4, encapsulated antibody plus saponin. b E (excellent) 5 5, P (poor) 5 1 on EUROP scale. c 1 5 lean, 5 5 fat. A,B Treatment group means with the same superscript within the same row are not different (P . 0.05).
g / kg) using clenbuterol and Moloney et al. (1995) (30 g / kg) using cimaterol administration. In the present study, the anti-idiotype response had no significant effect on carcass conformation. This may be partly explained by the fact that the antiidiotype response led to a large increase in carcass length. Conformation is assessed visually as carcass ‘blockiness’, i.e. the thickness of muscle and fat in relation to skeletal size. Thus, any increase in muscle size is unlikely to be detected by conformation assessment if skeletal size has increased. The lack of effect of the anti-idiotype response on leg length in our study is consistent with the findings of Moloney et al. (1995). Kidney and channel fat tended to be reduced
(P , 0.10) by beta-agonist administration in the study by Moloney et al. (1995). In the present study, there was no evidence of a reduction in this fat depot. This may reflect the fact that, in the present trial, the average weight of kidney and channel fat (when expressed on a per kg carcass weight basis) in the control group (17 g / kg) was much lower than the corresponding value (72 g / kg) in the study of Moloney et al. (1995). The linear fat measurements which were recorded in this trial are a direct measure of the subcutaneous fat content of the carcass. In the present study, no effects on linear fat measurements were observed, indicating that subcutaneous fat content was not affected. This agrees with the findings of Moloney et
A.F. Carson et al. / Livestock Production Science 68 (2001) 87 – 91
al. (1995) who reported that subcutaneous fat content was not significantly affected by beta-agonist administration in lambs.
5. Conclusions A murine monoclonal antibody against clenbuterol was demonstrated to generate an anti-idiotype response in lambs causing an increase in carcass length and dressing proportion.
References Carson, A.F., Moss, B.W., Steen, R.W.J., Kilpatrick, D.J., 1999. Effects of the percentage of Texel or Rouge de l’Ouest genes in lambs on carcass characteristics and meat quality. Anim. Sci. 69, 81–92. Elliott, C.T., Francis, K.S., McCaughey, W.J., 1994. Investigation of dissociation enhanced lanthanide fluorimmunoassay as an
91
alternative screening test for veterinary drug residues. The Analyst 119, 2565–2570. GENSTAT, 1993. GENSTAT 5 Release 3. Reference Manual. Clarendon Press, Oxford. Jurn, N.K., 1974. Towards a network theory of the immune system. Ann. Immunol. 125C, 373–389. Koohmaraie, M., Shackleford, S.D., Wheeler, T.L., 1996. Effects of a b-adrenergic agonist (L-644, 969) and male sex condition on muscle growth and meat quality of callipyge lambs. J. Anim. Sci. 74, 70–79. Moloney, A.P., Allen, P., McHugh, T.V., Quirke, J.F., 1995. Effects of cimaterol on Finnish-Landrace wether lambs. 1. Feed conversion efficiency, body composition and selected plasma hormone and metabolite concentrations. Livest. Prod. Sci. 42, 23–33. Rodgers, E.S., Elliott, C.T., Li Wan Po, A., Mackie, D.P., Scott, E.M., Kreuter, J., 1998. The potential of polymeric nanocapsules to increase the immunogenicity of the hapten clenbuterol. Food Agric. Immunol. 9, 159–166. Teh, C.Z., Wong, E., Lee, C.Y.G., 1984. Generation of monoclonal antibodies to human gonadotrophin by a facile cloning procedure. J. Appl. Biochem. 6, 48–55. Warriss, P.D., Kestin, S.C., Brown, S.N., 1989. The effect of beta-adrenergic agonists on carcass and meat quality in sheep. Anim. Prod. 48, 385–392.
Short communication
Active immunisation of lambs with a monoclonal antibody against clenbuterol A.F. Carson a
a,b ,
*, C.T. Elliott a,c , D.P. Mackie a,c , W.J. McCaughey a,c
Department of Agriculture and Rural Development for Northern Ireland, Hillsborough, Co. Down BT26 6 DR, Northern Ireland, UK b Agricultural Research Institute of Northern Ireland, Hillsborough, Co. Down BT26 6 DR, Northern Ireland, UK c Veterinary Sciences Division, Stoney Road, Belfast, BT3 4 SD, Northern Ireland, UK Received 14 August 1998; received in revised form 7 June 1999; accepted 28 April 2000
Abstract An experiment was undertaken with 50 Texel 3 Suffolk-Cheviot lambs (5468.8 days of age) to investigate the effects of active immunisation with a murine monoclonal antibody against clenbuterol on growth and carcass characteristics. Animals on treatments 1 and 2 each received 0.1 mg of clenbuterol antibody while animals on treatments 3 and 4 received 0.1 mg of antibody encapsulated within a synthetic polymer. Diethylaminoethyl (DEAE)-dextran was used as the adjuvant in treatments 1 and 3 and saponin in treatments 2 and 4. Control animals were immunised with saponin only. Four immunisations were given at 4-week intervals. Animals were slaughtered 4 weeks after the final immunisation. Each vaccine evoked a similar level of antibody response while the control group showed no titres. Lamb growth rate did not vary significantly between the vaccinated and control groups. Dressing proportion was higher (P , 0.05) in treatments 1 and 4 compared with the control group. Carcass length was greater (P , 0.001) in each of the vaccinated groups compared with the control group. None of the carcass fat depth measurements varied significantly between the control and vaccinated groups. 2001 Elsevier Science B.V. All rights reserved. Keywords: Clenbuterol; Anti-idiotype; Lambs; Carcass
1. Introduction Immunisation procedures can be used to generate antibody responses against a wide variety of antigens, including antibodies. Studies have shown that a proportion of anti-antibodies can react with the antigen combining site (idiotype) of the original
*Corresponding author. Tel.: 1 44-28-9268-2484; fax: 1 4428-9268-9594.
antibody and can therefore sometimes mimic (as an internal image) the action of the original antigen (Jurn, 1974). The objectives of the present study were firstly to determine if immunisation with a murine monoclonal antibody against the beta-agonist clenbuterol generated an anti-idiotype response in lambs, secondly to investigate the relative effectiveness of selected adjuvant formulations in generating an antibody response and thirdly to investigate the effects of anti-idiotype responses on growth and carcass characteristics in lambs.
0301-6226 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 00 )00206-2
88
A.F. Carson et al. / Livestock Production Science 68 (2001) 87 – 91
2. Materials and methods
2.1. Vaccine formulation and treatments A panel of monoclonal antibodies to clenbuterol was produced by the immunisation of a BALB / c mouse with a clenbuterol-transferrin conjugate. After five immunisations (at 3-week intervals) the mouse was killed and the spleen removed and fused with a mouse myeloma by the method of Teh et al. (1984). One of the clones (8B11) secreting clenbuterol antibody was selected (based on titre and cross reactivity profiles) for the production of ascites fluid for use in the present study. For each dose of treatment 1 vaccine, 2 ml of diethylaminoethyl (DEAE)-dextran (Sigma, Poole, UK) (2% solution) was added dropwise, while stirring, to 2 ml of clenbuterol antibody (0.05 mg / ml) at room temperature. For each dose of treatment 2 vaccine, 2 ml of saponin (‘Quil A’: Superfos, Vaalsgard, Denmark) (1 mg / ml) was added dropwise, while stirring, to 2 ml of clenbuterol antibody (0.05 mg / ml) at room temperature. For treatment 3 vaccine (encapsulated antigen plus DEAE-dextran), nanosphere preparation was achieved by emulsion polymerisation (Rodgers et al., 1998). For each vaccine dose, encapsulated antibody (0.1 mg) was diluted in saline (0.05 mg / ml) and to this solution was added 2 ml of DEAE-dextran (2% solution). For each dose of treatment 4 vaccine (encapsulated antigen plus saponin), 2 ml of saponin (1 mg / ml) was added dropwise, while stirring, to 2 ml of clenbuterol antibody (0.05 mg / ml) at room temperature. Control treatment animals received 2 ml of saponin (1 mg / ml).
2.2. Animals and treatments Fifty Texel 3 Suffolk-Cheviot lambs (n 5 19 rams, n 5 31 ewes) with an initial age of 5468.8 days and weight of 20.664.99 kg were allocated to five treatment groups. These groups were balanced for gender, age and live weight. Lambs were grazed with their mothers as a single group on perennial ryegrass-based swards. At approximately 90 days of age, all lambs were weaned and grazed together as a single group.
Four immunisations, administered by intramuscular injection, were given at 4-week intervals. Jugular blood samples (10 ml) were collected immediately prior to the first immunisation and at 14 days after each of the immunisations. The heparinised plasma was assayed for antibodies against the anti-clenbuterol antibody by means of a dissociation enhanced lanthanide fluorimmunoassay (DELFIA). Animals were weighed at 14-day intervals throughout the study and 4 weeks after the fourth immunisation animals were sent for slaughter. Carcasses were commercially graded for conformation (EUROP classification) and fat class (1 5 low fat cover; 5 5 high fat cover). Dressing proportion and linear carcass measurements were recorded as detailed by Carson et al. (1999).
2.3. Clenbuterol antibody assay A purified clenbuterol–human serum albumin conjugate was prepared as described by Elliott et al. (1994). This conjugate was labelled with europium (Elliott et al., 1994) and the resultant complex (CBLHSA-Eu 31 ) was stored in an equal volume of glycerol at 2 208C until required. Microtitre plates (Wallac, Turku, Finland) were coated with 100 ml of mouse anti-rabbit IgG (Sigma) diluted in 10 ml 50 mM carbonate / bicarbonate buffer pH 9.5. Plates were incubated at 378C for 2 h in a microtitre plate incubator / shaker. After a single wash with a 0.9% saline, 1% Tween 20 solution, excess reagent was tapped out onto clean tissue paper. In triplicate, a range of clenbuterol standards (0, 10, 25, 50, 125 and 1250 pg / well) prepared in distilled water were added to microtitre plate wells (25 ml / well). All other wells received 25 ml of distilled water. Into all wells was pipetted 50 ml of an anti-clenbuterol polyclonal antiserum raised in a rabbit, diluted to 1 / 20 000 in a 50 mM Tris–HCl buffer pH 7.4 containing 5 mg / ml BSA (assay buffer). All wells then received 50 ml of CBl-HSAEu 31 diluted 1 / 600 in assay buffer. To the wells of standards was then added 25 ml of new born calf serum (Gibco Ltd., Paisley, Scotland). Plasma samples taken from experimental animals were then added to the remaining wells (25 ml / well in triplicate). Plates were shaken gently at room tempera-
A.F. Carson et al. / Livestock Production Science 68 (2001) 87 – 91
ture for 2 h after which they were washed six times in wash solution. Into each well of the microtitre plates was added 100 ml of europium enhancement solution (Wallac). After a 10-min shake, plates were transferred into a time resolved fluorometer (Wallac 1234 reader) and the fluorescent intensity in each well measured at 630 nm. Standards were used to construct a spline fit standard curve and concentrations of anti-idiotype present in each sample were calculated and quantified in terms of clenbuterol equivalents (ng equiv).
2.4. Statistical analyses
89
Lamb growth rate (calculated by regression) did not vary significantly between the vaccinated and control groups (Table 2). However, dressing proportion was affected by immunisation. Dressing proportion was higher (P , 0.05) in treatments 1 and 4 than in the control group. Carcass length was greater (P , 0.001) in each of the vaccine treatments compared with the control group. None of the carcass fat parameters varied significantly between the control and vaccine treatments.
4. Discussion
The results were analysed by analysis of variance (Genstat, 1993) corresponding to the 5 (treatments) 3 2 (gender) factorial design of the experiment. Initial weight of the lambs was used as a covariate. Antibody responses at each time interval were analysed separately. As there were no significant treatment 3 gender interactions, only main effect means are presented in the tables.
3. Results There were no significant antibody responses generated by the vaccines after the first immunisation (Table 1). However, after the second immunisation, antibody responses (P , 0.05) were recorded in treatments 2 and 3 (vs. controls). After a fourth immunisation, antibody responses (P , 0.01) were recorded in all vaccine treatments.
In the present study, anti-idiotype antibodies to clenbuterol had no effect on lamb growth rate. The effects of beta-agonists on growth rate have been found to be variable. In some studies a significant increase in liveweight gain has been reported (Moloney et al., 1995), while in others, no effect has been observed (Koohmaraie et al., 1996). Moloney et al. (1995) suggested that the response to betaagonists may depend on the growth potential of the animals. The lamb genotype used in the present study may be classified as having a high growth potential and thus have a reduced capacity to respond to the anabolic effects of beta-agonists. Dressing proportion was increased by beta-agonist administration in some studies (Moloney et al., 1995). In the present study, the increase (42–49 g / kg) in dressing proportion in treatments 1 and 4 compared with the controls is in the order of magnitude reported by Warriss et al. (1989) (40
Table 1 Antibody responses to immunisation (ng equiv) Time interval b
PrePost Post Post Post a
first second third fourth
Control
ND 0.0 0.0 A 0.0 A 0.0 A
Treatment groups a
Gender
Significance of effects
1
2
3
4
S.E.D.
Males
Females
S.E.D.
Treatment
Gender
ND 0.1 0.5 AB 0.4 AB 0.8 B
ND 0.1 0.7 B 0.6 B 0.8 B
ND 0.0 0.7 B 0.4 AB 0.8 B
ND 0.0 0.5 AB 0.6 B 0.9 B
– 0.04 0.26 0.23 0.28
ND 0.1 0.4 0.4 0.5
ND 0.0 0.6 0.4 0.8
– 0.02 0.17 0.17 0.18
– NS ** * ***
– NS NS NS NS
1, antibody plus DEAE-dextran; 2, antibody plus saponin; 3, encapsulated antibody plus DEAE-dextran; 4, encapsulated antibody plus saponin. b Blood samples taken immediately prior to first immunisation and 14 days after each immunisation. A,B Treatment group means with the same superscript within the same row are not different (P . 0.05). ND, not detectable.
A.F. Carson et al. / Livestock Production Science 68 (2001) 87 – 91
90
Table 2 Carcass characteristics in the treatment groups Control
Treatment groups a
Gender
Significance of effects
1
2
3
4
S.E.D.
Males
Females
S.E.D.
Treatment
Gender
Growth rate (g/day) Dressing proportion (g/kg) Carcass weight (kg) Conformation classification b Fat classification c Kidney 1 Channel fat (g) Kidney weight (g) Kidney weight (g/kg half carcass weight)
224 488 A 22.1 3.4 4.3 382 141 3.1
241 530 B 23.7 3.5 3.6 505 128 2.7
218 523 AB 23.7 3.5 4.0 577 129 2.4
211 496 AB 23.2 3.5 4.2 383 129 3.2
218 537 B 23.2 3.5 4.0 466 137 2.4
15.3 20.9 1.20 0.43 0.33 95.9 17.0 0.49
241 504 24.0 3.4 4.2 450 130 2.5
205 525 22.4 3.5 3.9 475 136 3.1
9.6 13.1 0.75 0.27 0.21 60.0 10.7 0.31
NS * NS NS NS NS NS NS
*** * * NS NS NS NS *
Carcass linear measurements (mm) Carcass length Width of barrel Width of shoulder Leg length Circumference of buttocks
519 A 246 220 284 462
570 B 251 222 283 484
569 B 248 226 275 487
548 B 251 218 268 491
559 B 251 223 271 485
13.9 10.5 8.3 7.8 15.1
556 252 224 276 486
550 247 219 276 478
8.7 6.6 5.2 4.9 9.4
*** NS NS NS NS
NS NS NS NS NS
3.4 2.4
3.5 2.9
3.9 3.9
2.7 2.7
3.1 3.5
1.10 0.70
3.2 3.2
3.4 3.0
0.69 0.44
NS NS
NS NS
6.1 6.8 6.5 12.9
6.6 7.7 7.3 13.7
7.8 8.2 7.7 13.6
6.1 6.8 6.3 11.3
7.4 8.2 8.4 12.9
1.43 1.38 1.25 1.77
7.3 7.2 7.0 12.0
6.3 7.9 7.6 13.8
0.90 0.86 0.78 1.11
NS NS NS NS
NS NS NS NS
Linear fat measurements (mm) Fat depth over Longissimus dorsi 35 mm from midline 50 mm from midline Fat depth at loin (mm) 35 mm from midline Above Gluteus medius At Obliqus internus abdominis Tissue depth (mm) a
1, antibody plus DEAE-dextran; 2, antibody plus saponin; 3, encapsulated antibody plus DEAE-dextran; 4, encapsulated antibody plus saponin. b E (excellent) 5 5, P (poor) 5 1 on EUROP scale. c 1 5 lean, 5 5 fat. A,B Treatment group means with the same superscript within the same row are not different (P . 0.05).
g / kg) using clenbuterol and Moloney et al. (1995) (30 g / kg) using cimaterol administration. In the present study, the anti-idiotype response had no significant effect on carcass conformation. This may be partly explained by the fact that the antiidiotype response led to a large increase in carcass length. Conformation is assessed visually as carcass ‘blockiness’, i.e. the thickness of muscle and fat in relation to skeletal size. Thus, any increase in muscle size is unlikely to be detected by conformation assessment if skeletal size has increased. The lack of effect of the anti-idiotype response on leg length in our study is consistent with the findings of Moloney et al. (1995). Kidney and channel fat tended to be reduced
(P , 0.10) by beta-agonist administration in the study by Moloney et al. (1995). In the present study, there was no evidence of a reduction in this fat depot. This may reflect the fact that, in the present trial, the average weight of kidney and channel fat (when expressed on a per kg carcass weight basis) in the control group (17 g / kg) was much lower than the corresponding value (72 g / kg) in the study of Moloney et al. (1995). The linear fat measurements which were recorded in this trial are a direct measure of the subcutaneous fat content of the carcass. In the present study, no effects on linear fat measurements were observed, indicating that subcutaneous fat content was not affected. This agrees with the findings of Moloney et
A.F. Carson et al. / Livestock Production Science 68 (2001) 87 – 91
al. (1995) who reported that subcutaneous fat content was not significantly affected by beta-agonist administration in lambs.
5. Conclusions A murine monoclonal antibody against clenbuterol was demonstrated to generate an anti-idiotype response in lambs causing an increase in carcass length and dressing proportion.
References Carson, A.F., Moss, B.W., Steen, R.W.J., Kilpatrick, D.J., 1999. Effects of the percentage of Texel or Rouge de l’Ouest genes in lambs on carcass characteristics and meat quality. Anim. Sci. 69, 81–92. Elliott, C.T., Francis, K.S., McCaughey, W.J., 1994. Investigation of dissociation enhanced lanthanide fluorimmunoassay as an
91
alternative screening test for veterinary drug residues. The Analyst 119, 2565–2570. GENSTAT, 1993. GENSTAT 5 Release 3. Reference Manual. Clarendon Press, Oxford. Jurn, N.K., 1974. Towards a network theory of the immune system. Ann. Immunol. 125C, 373–389. Koohmaraie, M., Shackleford, S.D., Wheeler, T.L., 1996. Effects of a b-adrenergic agonist (L-644, 969) and male sex condition on muscle growth and meat quality of callipyge lambs. J. Anim. Sci. 74, 70–79. Moloney, A.P., Allen, P., McHugh, T.V., Quirke, J.F., 1995. Effects of cimaterol on Finnish-Landrace wether lambs. 1. Feed conversion efficiency, body composition and selected plasma hormone and metabolite concentrations. Livest. Prod. Sci. 42, 23–33. Rodgers, E.S., Elliott, C.T., Li Wan Po, A., Mackie, D.P., Scott, E.M., Kreuter, J., 1998. The potential of polymeric nanocapsules to increase the immunogenicity of the hapten clenbuterol. Food Agric. Immunol. 9, 159–166. Teh, C.Z., Wong, E., Lee, C.Y.G., 1984. Generation of monoclonal antibodies to human gonadotrophin by a facile cloning procedure. J. Appl. Biochem. 6, 48–55. Warriss, P.D., Kestin, S.C., Brown, S.N., 1989. The effect of beta-adrenergic agonists on carcass and meat quality in sheep. Anim. Prod. 48, 385–392.