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Short communication

Active immunisation of lambs with a monoclonal antibody

against clenbuterol

a,b ,

_*

a,c a,c a,c

A.F. Carson

, C.T. Elliott

, D.P. Mackie

, W.J. McCaughey

a

Department of Agriculture and Rural Development for Northern Ireland, Hillsborough, Co. Down BT26 6DR, Northern Ireland, UK

b

Agricultural Research Institute of Northern Ireland, Hillsborough, Co. Down BT26 6DR, Northern Ireland, UK

c

Veterinary Sciences Division, Stoney Road, Belfast, BT3 4SD, 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 Texel3Suffolk-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.

Keywords: Clenbuterol; Anti-idiotype; Lambs; Carcass

1. Introduction antibody and can therefore sometimes mimic (as an

internal image) the action of the original antigen Immunisation procedures can be used to generate (Jurn, 1974). The objectives of the present study antibody responses against a wide variety of an- were firstly to determine if immunisation with a tigens, including antibodies. Studies have shown that murine monoclonal antibody against the beta-agonist a proportion of anti-antibodies can react with the clenbuterol generated an anti-idiotype response in antigen combining site (idiotype) of the original lambs, secondly to investigate the relative effective-ness of selected adjuvant formulations in generating an antibody response and thirdly to investigate the effects of anti-idiotype responses on growth and *Corresponding author. Tel.: 144-28-9268-2484; fax: 1

44-28-9268-9594. carcass characteristics in lambs.

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2. Materials and methods Four immunisations, administered by intramuscu-lar injection, were given at 4-week intervals. Juguintramuscu-lar blood samples (10 ml) were collected immediately 2.1. Vaccine formulation and treatments

prior to the first immunisation and at 14 days after each of the immunisations. The heparinised plasma A panel of monoclonal antibodies to clenbuterol

was assayed for antibodies against the anti-clen-was produced by the immunisation of a BALB / c

buterol antibody by means of a dissociation en-mouse with a clenbuterol-transferrin conjugate. After

hanced lanthanide fluorimmunoassay (DELFIA). five immunisations (at 3-week intervals) the mouse

Animals were weighed at 14-day intervals was killed and the spleen removed and fused with a

throughout the study and 4 weeks after the fourth mouse myeloma by the method of Teh et al. (1984).

immunisation animals were sent for slaughter. Car-One of the clones (8B11) secreting clenbuterol

casses were commercially graded for conformation antibody was selected (based on titre and cross

(EUROP classification) and fat class (15low fat reactivity profiles) for the production of ascites fluid

cover; 55high fat cover). Dressing proportion and for use in the present study.

linear carcass measurements were recorded as de-For each dose of treatment 1 vaccine, 2 ml of

tailed by Carson et al. (1999). diethylaminoethyl (DEAE)-dextran (Sigma, Poole,

UK) (2% solution) was added dropwise, while stirring, to 2 ml of clenbuterol antibody (0.05 mg /

2.3. Clenbuterol antibody assay ml) at room temperature. For each dose of treatment

2 vaccine, 2 ml of saponin (‘Quil A’: Superfos,

A purified clenbuterol–human serum albumin Vaalsgard, Denmark) (1 mg / ml) was added

drop-conjugate was prepared as described by Elliott et al. wise, while stirring, to 2 ml of clenbuterol antibody

(1994). This conjugate was labelled with europium (0.05 mg / ml) at room temperature. For treatment 3

(Elliott et al., 1994) and the resultant complex (CBL-vaccine (encapsulated antigen plus DEAE-dextran),

31

HSA-Eu ) was stored in an equal volume of nanosphere preparation was achieved by emulsion

glycerol at 2208C until required. polymerisation (Rodgers et al., 1998). For each

Microtitre plates (Wallac, Turku, Finland) were vaccine dose, encapsulated antibody (0.1 mg) was

coated with 100ml of mouse anti-rabbit IgG (Sigma) diluted in saline (0.05 mg / ml) and to this solution

diluted in 10 ml 50 mM carbonate / bicarbonate was added 2 ml of DEAE-dextran (2% solution). For

buffer pH 9.5. Plates were incubated at 378C for 2 h each dose of treatment 4 vaccine (encapsulated

in a microtitre plate incubator / shaker. After a single antigen plus saponin), 2 ml of saponin (1 mg / ml)

wash with a 0.9% saline, 1% Tween 20 solution, was added dropwise, while stirring, to 2 ml of

excess reagent was tapped out onto clean tissue clenbuterol antibody (0.05 mg / ml) at room

tempera-paper. In triplicate, a range of clenbuterol standards ture. Control treatment animals received 2 ml of

(0, 10, 25, 50, 125 and 1250 pg / well) prepared in saponin (1 mg / ml).

distilled water were added to microtitre plate wells (25 ml / well). All other wells received 25 ml of 2.2. Animals and treatments distilled water. Into all wells was pipetted 50 ml of an anti-clenbuterol polyclonal antiserum raised in a Fifty Texel3Suffolk-Cheviot lambs (n519 rams, rabbit, diluted to 1 / 20 000 in a 50 mM Tris–HCl n531 ewes) with an initial age of 5468.8 days and buffer pH 7.4 containing 5 mg / ml BSA (assay weight of 20.664.99 kg were allocated to five buffer). All wells then received 50 ml of

CBl-HSA-31

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tempera-ture for 2 h after which they were washed six times Lamb growth rate (calculated by regression) did in wash solution. Into each well of the microtitre not vary significantly between the vaccinated and plates was added 100 ml of europium enhancement control groups (Table 2). However, dressing propor-solution (Wallac). After a 10-min shake, plates were tion was affected by immunisation. Dressing propor-transferred into a time resolved fluorometer (Wallac tion was higher (P,0.05) in treatments 1 and 4 than 1234 reader) and the fluorescent intensity in each in the control group. Carcass length was greater well measured at 630 nm. Standards were used to (P,0.001) in each of the vaccine treatments com-construct a spline fit standard curve and concen- pared with the control group. None of the carcass fat trations of anti-idiotype present in each sample were parameters varied significantly between the control calculated and quantified in terms of clenbuterol and vaccine treatments.

equivalents (ng equiv).

2.4. Statistical analyses

4. Discussion

The results were analysed by analysis of variance

In the present study, anti-idiotype antibodies to (Genstat, 1993) corresponding to the 5

clenbuterol had no effect on lamb growth rate. The (treatments)32 (gender) factorial design of the

effects of beta-agonists on growth rate have been experiment. Initial weight of the lambs was used as a

found to be variable. In some studies a significant covariate. Antibody responses at each time interval

increase in liveweight gain has been reported were analysed separately. As there were no

signifi-(Moloney et al., 1995), while in others, no effect has cant treatment3gender interactions, only main

ef-been observed (Koohmaraie et al., 1996). Moloney fect means are presented in the tables.

et al. (1995) suggested that the response to beta-agonists may depend on the growth potential of the animals. The lamb genotype used in the present

3. Results study may be classified as having a high growth

potential and thus have a reduced capacity to respond There were no significant antibody responses to the anabolic effects of beta-agonists.

generated by the vaccines after the first immunisation Dressing proportion was increased by beta-agonist (Table 1). However, after the second immunisation, administration in some studies (Moloney et al., antibody responses (P,0.05) were recorded in 1995). In the present study, the increase (42–49 treatments 2 and 3 (vs. controls). After a fourth g / kg) in dressing proportion in treatments 1 and 4 immunisation, antibody responses (P,0.01) were compared with the controls is in the order of recorded in all vaccine treatments. magnitude reported by Warriss et al. (1989) (40

Table 1

Antibody responses to immunisation (ng equiv)

b a

Time interval Control Treatment groups Gender Significance of effects

1 2 3 4 S.E.D. Males Females S.E.D. Treatment Gender

Pre- ND ND ND ND ND – ND ND – – –

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

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Table 2

Carcass characteristics in the treatment groups

a

Control Treatment groups Gender Significance of effects

1 2 3 4 S.E.D. Males Females S.E.D. Treatment Gender

Growth rate (g / day) 224 241 218 211 218 15.3 241 205 9.6 NS ***

A B AB AB B

Dressing proportion (g / kg) 488 530 523 496 537 20.9 504 525 13.1 * *

Carcass weight (kg) 22.1 23.7 23.7 23.2 23.2 1.20 24.0 22.4 0.75 NS *

b

Conformation classification 3.4 3.5 3.5 3.5 3.5 0.43 3.4 3.5 0.27 NS NS

c

Fat classification 4.3 3.6 4.0 4.2 4.0 0.33 4.2 3.9 0.21 NS NS

Kidney1Channel fat (g) 382 505 577 383 466 95.9 450 475 60.0 NS NS

Kidney weight (g) 141 128 129 129 137 17.0 130 136 10.7 NS NS

Kidney weight (g / kg half carcass weight) 3.1 2.7 2.4 3.2 2.4 0.49 2.5 3.1 0.31 NS *

Carcass linear measurements (mm)

A B B B B

Carcass length 519 570 569 548 559 13.9 556 550 8.7 *** NS

Width of barrel 246 251 248 251 251 10.5 252 247 6.6 NS NS

Width of shoulder 220 222 226 218 223 8.3 224 219 5.2 NS NS

Leg length 284 283 275 268 271 7.8 276 276 4.9 NS NS

Circumference of buttocks 462 484 487 491 485 15.1 486 478 9.4 NS NS

Linear fat measurements (mm) Fat depth over Longissimus dorsi

35 mm from midline 3.4 3.5 3.9 2.7 3.1 1.10 3.2 3.4 0.69 NS NS

Fat depth at loin (mm)

Above Gluteus medius 6.8 7.7 8.2 6.8 8.2 1.38 7.2 7.9 0.86 NS NS

At Obliqus internus abdominis 6.5 7.3 7.7 6.3 8.4 1.25 7.0 7.6 0.78 NS NS

Tissue depth (mm) 12.9 13.7 13.6 11.3 12.9 1.77 12.0 13.8 1.11 NS NS

a

1, antibody plus DEAE-dextran; 2, antibody plus saponin; 3, encapsulated antibody plus DEAE-dextran; 4, encapsulated antibody plus saponin.

b

E (excellent)55, P (poor)51 on EUROP scale.

c

15lean, 55fat.

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) (P,0.10) by beta-agonist administration in the (30 g / kg) using cimaterol administration. study by Moloney et al. (1995). In the present study, In the present study, the anti-idiotype response had there was no evidence of a reduction in this fat no significant effect on carcass conformation. This depot. This may reflect the fact that, in the present may be partly explained by the fact that the anti- trial, the average weight of kidney and channel fat idiotype response led to a large increase in carcass (when expressed on a per kg carcass weight basis) in length. Conformation is assessed visually as carcass the control group (17 g / kg) was much lower than ‘blockiness’, i.e. the thickness of muscle and fat in the corresponding value (72 g / kg) in the study of relation to skeletal size. Thus, any increase in muscle Moloney et al. (1995).

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alternative screening test for veterinary drug residues. The al. (1995) who reported that subcutaneous fat content

Analyst 119, 2565–2570. was not significantly affected by beta-agonist

ad-GENSTAT, 1993. GENSTAT 5 Release 3. Reference Manual. ministration in lambs. _{Clarendon Press, Oxford.}

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Koohmaraie, M., Shackleford, S.D., Wheeler, T.L., 1996. Effects 5. Conclusions _{of a}_b_{-adrenergic agonist (L-644, 969) and male sex condition}

on muscle growth and meat quality of callipyge lambs. J. A murine monoclonal antibody against clenbuterol Anim. Sci. 74, 70–79.

Moloney, A.P., Allen, P., McHugh, T.V., Quirke, J.F., 1995. was demonstrated to generate an anti-idiotype

re-Effects of cimaterol on Finnish-Landrace wether lambs. 1. Feed sponse in lambs causing an increase in carcass length

conversion efficiency, body composition and selected plasma and dressing proportion. _{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 nanocap-References _{sules to increase the immunogenicity of the hapten clenbuterol.}

Food Agric. Immunol. 9, 159–166.

Carson, A.F., Moss, B.W., Steen, R.W.J., Kilpatrick, D.J., 1999. Teh, C.Z., Wong, E., Lee, C.Y.G., 1984. Generation of monoclonal Effects of the percentage of Texel or Rouge de l’Ouest genes in antibodies to human gonadotrophin by a facile cloning pro-lambs on carcass characteristics and meat quality. Anim. Sci. cedure. J. Appl. Biochem. 6, 48–55.

69, 81–92. Warriss, P.D., Kestin, S.C., Brown, S.N., 1989. The effect of Elliott, C.T., Francis, K.S., McCaughey, W.J., 1994. Investigation beta-adrenergic agonists on carcass and meat quality in sheep.