- Email: [email protected]
Serum Antibody Responses to a Clostridium perfringens Epsilon Toxoid Vaccine in Goats
Anaerobe (1999) 5, 287±289 Article No. anae.1999.0292
VETERINARY MICROBIOLOGY (ORAL PRESENTATION)
Serum Antibody Responses to a Clostridium perfringens EpsilonToxoid Vaccine in Goats F.A. Uzal* and W.R. Kelly School of Veterinary Science and Animal Production, The University of Queensland, Brisbane Qld 4072, Australia
Key Words: Clostridium perfringens, Epsilon toxoid vaccine, goats
Introduction Enterotoxaemia caused by Clostridium perfringens type D (pulpy kidney disease) is a common disease of sheep and goats [1,2]. Commercial sheep enterotoxaemia vaccines consist of an epsilon toxoid [3] adsorbed onto an adjuvant (usually aluminium hydroxide), and because no enterotoxaemia vaccines are specially manufactured for goats, these sheep vaccines are also generally used in that species [4]. There are abundant references about immunity to enterotoxaemia in sheep [5±8] and there is no doubt that sheep can be fully protected against this disease by vaccination [9]. However, data on antibody titres and local reactions in goats after enterotoxaemia vaccination are scanty and contradictory [4,10]. We present here a vaccination trial with a commercial vaccine in goats in order to measure
* Corresponding author: F.U. Uzal, Tel.: 61 7 3365 2881; Fax: 61 7 3365 1355. E-mail: [email protected]
1075±9964/99/030287 + 03 $30.00/0
their antibody responses and local reactions to different vaccination regimes.
Materials and Methods Twenty-nine Angora goat kids, 3±4 months old, were randomly allocated to four groups (1±4); eight animals each to groups 1 and 2, nine to group 3 and 4 animals to group 4. At the beginning of the experiment (day 0), animals in groups 1, 2 and 3 received 1 dose of vaccine. Animals in groups 2 and 3 received a booster of the same vaccine at days 28 and 42 after the first vaccination, respectively. Group 4 remained as an unvaccinated control. All the animals were bled at the beginning of the trial (day 0) and on days 17, 28, 42, 59, 70, 86, 98 and 128. The sera were processed by an ELISA technique for epsilon toxin antibodies as previously described [11]. A combined caseous lymphadenitis, tetanus and enterotoxaemia vaccine (``Glanvac 3'', CSL Limited, Melbourne, Australia) was used. It was administered subcutaneously in the internal side of the right (first # 1999 Academic Press
288
F.A. Uzal and W.R. Kelly
dose) or left (second dose) thigh. New disposable, 21 gauge, one-inch needles were used for each animal and the skin at the injection site was swabbed with 70% ethanol before injection. Before the first vaccination and on days 17, 28, 42, 59 and 70, the thickness of the skin fold at the site of the first injection was measured using Vernier callipers. Based on serum epsilon antitoxin titres within groups, each goat was placed into one of the following two categories: (a) low antibody titres (less than 0.25 IU epsilon antitoxin per ml of serum) or (b) high antibody titres (more than 0.25 IU epsilon antitoxin per ml of serum). The distribution of these categories for each group were compared at the 9 bleeding occasions using a chi-square test of dependence [12]. The titres were all transformed to a Normal Distribution using a log transformation and analysed using one-way analysis of variance to determine if there was a significant effect of group or time.
Results A great majority of the animals in three groups (groups 2, 3 and 4) had serum antibodies below the protective level at day 98 after the beginning of the trial, and most animals in group 1 had serum antibodies below the protective level at day 128. An increase in the antibody titre occurred in groups 1, 2 and 3 after each vaccination. The titers were below the putative level (0.25 IU/mL) in groups 1, 2 and 3 by days 42, 70 and 86, respectively. Titers were below the protection level in all occasions for group 4. The status (protected or unprotected) of the four groups before and after vaccination is shown in Table 1. The thickness of the skin before vaccination (day 0) in the test animals (groups 1 to 3) ranged between 0.17 cm and 0.35 cm, the median value being 0.22 cm, while in the control group these values ranged between 0.18 cm and 0.32 cm, with a median value of 0.20 cm. In the test animals (groups 1 to 3) when the injection site of the vaccine was measured 17 days after the first vaccination the thickness of the skin ranged between 0.30 cm and 2.05 cm (median Table 1. Status (protected or unprotected) of four groups of animals (average of each group) before vaccination and at different intervals thereafter Days Group 1 2 3 4
0
17
28
42
59
70
86
98
128
U* U* U* U
P P P U
P U* U U
U P U* U
U P P U
U U P U
U U U U
U U U U
U U U U
U: unprotected, P: protected, * : vaccination.
0.70 cm). In most cases, a moderately firm lump was observed at the injection site. In three cases, a caseous material could be squeezed from these lumps. The thickness of the skin of the test animals had diminished by day 28 (range 0.25 cm to 0.90 cm; median 0.45 cm) and remained constant thereafter. No changes were detected in the thickness of the skin of the control group throughout the experimental period.
Discussion It has been recommended that to maintain a protective level of immunity, goats should be vaccinated every 3 to 4 months following an initial double vaccination, 3 to 6 weeks apart [2]. Although the antibody level threshold for protection against enterotoxaemia has not yet been definitely established in goats, it was found in a trial with a small number of animals that goats with antibody titres between 2.45 and 230.00 IU/mL of epsilon antitoxin were fully protected against the local (intestinal) and systemic (cerebral and pulmonary) effects of an experimental challenge with C. perfringens culture supernatants, while animals with antibody titres between 0.22 and 1.52 IU/mL developed a slight diarrhoea [13]. Blackwell et al. arbitrarily classified goats with antibody levels of less than 0.10 IU/mL as unprotected, 0.10 to 1.00 IU/mL as at risk, and more than 1.00 IU/mL as protected [4]. In sheep, different authors consider that the level of protection varies between 0.10 IU/ml and 0.30 IU/mL [5,9,14]. In this study, it was decided to adopt a relatively conservative approach and classify a titre of 0.25 IU/mL as representing the threshold for protection. If this value is indeed the case, the results presented here support the idea suggested by Shanks (1949) [10] and Green (1987) [15], that protective antibody levels in goats are of short duration and that animals must, therefore, be re-vaccinated between 2 and 3 months after the first double vaccination, and at similar intervals thereafter [2]. This is cumbersome for goat production on a commercial basis. The use of different adjuvants for a vaccine that confers long lasting immunity to goats is presently being studied by the authors. A high incidence (up to 100%) of local reactions following vaccination of goats was observed by Blackwell et al. [4] and also by Green et al. (1987) [15]. They consequently suggested that such immunogens should be administered at sites distant from regional lymph nodes in order to avoid confusion with the lesions of caseous lymphadenitis. However, similar reactions were observed in 100% of sheep vaccinated with three different commercial vaccines against clostridial diseases [15], which seems to
Serum antibody responses indicate that these undesirable reactions are not restricted to goats. In the present trial local reactions were always mild and disappeared almost completely about 1 month after vaccination. However, new, sterile needles were used for each animal and the skin at the injection site was disinfected with alcohol, a procedure not commonly followed in goat practice.
References 1. Niilo L. (1980) Clostridium perfringens in animal disease: a review of current knowledge. Can Vet J 21: 141±148 2. Smith M.C. and Sherman D.M. (1994) Goat Medicine. Lea and Febiger, Pennsylvania, USA. pp. 620 3. British Pharmacopoeia (Veterinary) (1993) HMSO. London, England. pp. 118 4. Blackwell T.E., Butler D.G. and Bell J.A. (1983) Enterotoxaemia in the goat: the humoral response and local tissue reaction following vaccination with two different bacterin-toxoids. Can J Comparative Pathol Med 47: 127±132 5. Sterne M., Batty I.I., Thomson A. and Robertson J.M. (1962) Immunisation of sheep with multi-component clostridial vaccines. Vet Record 74: 909±913
289
6. Jansen B.C., Visser F. and Knoetze P.C.C. (1965) Some practical considerations on the protection of sheep against pulpy kidney disease. J South African Vet Med Assoc 6: 365±366 7. Jansen B.C. (1967) The prevention of enterotoxaemia (pulpy kidney disease) by vaccination. Bull Int Office Epizootics 67: 1539±1567 8. Oxer D.T., Minty D.W. and Liefman C.E. (1971) Vaccination trials in sheep with clostridial vaccines with special reference to passively acquired Clostridium welchii type D antitoxin in lambs. Austral Vet J 47: 134±140 9. Jansen B.C. (1960) The Experimental reproduction of pulpy kidney disease. J South African Vet Med Assoc 31: 205±208 10. Shanks P.L. (1949) Enterotoxaemia in goats. Vet Record 61: 262±264 11. Uzal F.A., Nielsen K. and Kelly W.R. (1997) Competitive and indirect ELISA detection of Clostridium perfringens type D epsilon antitoxin in serum of goats. Vet Microbiol 24 51: 223±231 12. Moore D.S. and McCabe G.P. (1993) Introduction to the Practice of Statistics, 2nd ed. W.H. Freeman and Company, New York, USA. pp. 713±789 13. Uzal F.A. and Kelly W.R. (1998) Protection of goats against experimental enterotoxaemia by vaccination with Clostridium perfringens type D epsilon toxoid. Vet Record 24 142: 722±725 14. Smith L.D.S. and Marsh H. (1953) The immunization of young lambs against enterotoxaemia. Am J Vet Res 14: 408±410 15. Green D.S., Green M.J., Hillyer M.H. and Morgan K.L. (1987) Injection site reactions and antibody responses in sheep and goats after the use of a multivalent clostridial vaccine. Vet Record 120: 435±439
VETERINARY MICROBIOLOGY (ORAL PRESENTATION)
Serum Antibody Responses to a Clostridium perfringens EpsilonToxoid Vaccine in Goats F.A. Uzal* and W.R. Kelly School of Veterinary Science and Animal Production, The University of Queensland, Brisbane Qld 4072, Australia
Key Words: Clostridium perfringens, Epsilon toxoid vaccine, goats
Introduction Enterotoxaemia caused by Clostridium perfringens type D (pulpy kidney disease) is a common disease of sheep and goats [1,2]. Commercial sheep enterotoxaemia vaccines consist of an epsilon toxoid [3] adsorbed onto an adjuvant (usually aluminium hydroxide), and because no enterotoxaemia vaccines are specially manufactured for goats, these sheep vaccines are also generally used in that species [4]. There are abundant references about immunity to enterotoxaemia in sheep [5±8] and there is no doubt that sheep can be fully protected against this disease by vaccination [9]. However, data on antibody titres and local reactions in goats after enterotoxaemia vaccination are scanty and contradictory [4,10]. We present here a vaccination trial with a commercial vaccine in goats in order to measure
* Corresponding author: F.U. Uzal, Tel.: 61 7 3365 2881; Fax: 61 7 3365 1355. E-mail: [email protected]
1075±9964/99/030287 + 03 $30.00/0
their antibody responses and local reactions to different vaccination regimes.
Materials and Methods Twenty-nine Angora goat kids, 3±4 months old, were randomly allocated to four groups (1±4); eight animals each to groups 1 and 2, nine to group 3 and 4 animals to group 4. At the beginning of the experiment (day 0), animals in groups 1, 2 and 3 received 1 dose of vaccine. Animals in groups 2 and 3 received a booster of the same vaccine at days 28 and 42 after the first vaccination, respectively. Group 4 remained as an unvaccinated control. All the animals were bled at the beginning of the trial (day 0) and on days 17, 28, 42, 59, 70, 86, 98 and 128. The sera were processed by an ELISA technique for epsilon toxin antibodies as previously described [11]. A combined caseous lymphadenitis, tetanus and enterotoxaemia vaccine (``Glanvac 3'', CSL Limited, Melbourne, Australia) was used. It was administered subcutaneously in the internal side of the right (first # 1999 Academic Press
288
F.A. Uzal and W.R. Kelly
dose) or left (second dose) thigh. New disposable, 21 gauge, one-inch needles were used for each animal and the skin at the injection site was swabbed with 70% ethanol before injection. Before the first vaccination and on days 17, 28, 42, 59 and 70, the thickness of the skin fold at the site of the first injection was measured using Vernier callipers. Based on serum epsilon antitoxin titres within groups, each goat was placed into one of the following two categories: (a) low antibody titres (less than 0.25 IU epsilon antitoxin per ml of serum) or (b) high antibody titres (more than 0.25 IU epsilon antitoxin per ml of serum). The distribution of these categories for each group were compared at the 9 bleeding occasions using a chi-square test of dependence [12]. The titres were all transformed to a Normal Distribution using a log transformation and analysed using one-way analysis of variance to determine if there was a significant effect of group or time.
Results A great majority of the animals in three groups (groups 2, 3 and 4) had serum antibodies below the protective level at day 98 after the beginning of the trial, and most animals in group 1 had serum antibodies below the protective level at day 128. An increase in the antibody titre occurred in groups 1, 2 and 3 after each vaccination. The titers were below the putative level (0.25 IU/mL) in groups 1, 2 and 3 by days 42, 70 and 86, respectively. Titers were below the protection level in all occasions for group 4. The status (protected or unprotected) of the four groups before and after vaccination is shown in Table 1. The thickness of the skin before vaccination (day 0) in the test animals (groups 1 to 3) ranged between 0.17 cm and 0.35 cm, the median value being 0.22 cm, while in the control group these values ranged between 0.18 cm and 0.32 cm, with a median value of 0.20 cm. In the test animals (groups 1 to 3) when the injection site of the vaccine was measured 17 days after the first vaccination the thickness of the skin ranged between 0.30 cm and 2.05 cm (median Table 1. Status (protected or unprotected) of four groups of animals (average of each group) before vaccination and at different intervals thereafter Days Group 1 2 3 4
0
17
28
42
59
70
86
98
128
U* U* U* U
P P P U
P U* U U
U P U* U
U P P U
U U P U
U U U U
U U U U
U U U U
U: unprotected, P: protected, * : vaccination.
0.70 cm). In most cases, a moderately firm lump was observed at the injection site. In three cases, a caseous material could be squeezed from these lumps. The thickness of the skin of the test animals had diminished by day 28 (range 0.25 cm to 0.90 cm; median 0.45 cm) and remained constant thereafter. No changes were detected in the thickness of the skin of the control group throughout the experimental period.
Discussion It has been recommended that to maintain a protective level of immunity, goats should be vaccinated every 3 to 4 months following an initial double vaccination, 3 to 6 weeks apart [2]. Although the antibody level threshold for protection against enterotoxaemia has not yet been definitely established in goats, it was found in a trial with a small number of animals that goats with antibody titres between 2.45 and 230.00 IU/mL of epsilon antitoxin were fully protected against the local (intestinal) and systemic (cerebral and pulmonary) effects of an experimental challenge with C. perfringens culture supernatants, while animals with antibody titres between 0.22 and 1.52 IU/mL developed a slight diarrhoea [13]. Blackwell et al. arbitrarily classified goats with antibody levels of less than 0.10 IU/mL as unprotected, 0.10 to 1.00 IU/mL as at risk, and more than 1.00 IU/mL as protected [4]. In sheep, different authors consider that the level of protection varies between 0.10 IU/ml and 0.30 IU/mL [5,9,14]. In this study, it was decided to adopt a relatively conservative approach and classify a titre of 0.25 IU/mL as representing the threshold for protection. If this value is indeed the case, the results presented here support the idea suggested by Shanks (1949) [10] and Green (1987) [15], that protective antibody levels in goats are of short duration and that animals must, therefore, be re-vaccinated between 2 and 3 months after the first double vaccination, and at similar intervals thereafter [2]. This is cumbersome for goat production on a commercial basis. The use of different adjuvants for a vaccine that confers long lasting immunity to goats is presently being studied by the authors. A high incidence (up to 100%) of local reactions following vaccination of goats was observed by Blackwell et al. [4] and also by Green et al. (1987) [15]. They consequently suggested that such immunogens should be administered at sites distant from regional lymph nodes in order to avoid confusion with the lesions of caseous lymphadenitis. However, similar reactions were observed in 100% of sheep vaccinated with three different commercial vaccines against clostridial diseases [15], which seems to
Serum antibody responses indicate that these undesirable reactions are not restricted to goats. In the present trial local reactions were always mild and disappeared almost completely about 1 month after vaccination. However, new, sterile needles were used for each animal and the skin at the injection site was disinfected with alcohol, a procedure not commonly followed in goat practice.
References 1. Niilo L. (1980) Clostridium perfringens in animal disease: a review of current knowledge. Can Vet J 21: 141±148 2. Smith M.C. and Sherman D.M. (1994) Goat Medicine. Lea and Febiger, Pennsylvania, USA. pp. 620 3. British Pharmacopoeia (Veterinary) (1993) HMSO. London, England. pp. 118 4. Blackwell T.E., Butler D.G. and Bell J.A. (1983) Enterotoxaemia in the goat: the humoral response and local tissue reaction following vaccination with two different bacterin-toxoids. Can J Comparative Pathol Med 47: 127±132 5. Sterne M., Batty I.I., Thomson A. and Robertson J.M. (1962) Immunisation of sheep with multi-component clostridial vaccines. Vet Record 74: 909±913
289
6. Jansen B.C., Visser F. and Knoetze P.C.C. (1965) Some practical considerations on the protection of sheep against pulpy kidney disease. J South African Vet Med Assoc 6: 365±366 7. Jansen B.C. (1967) The prevention of enterotoxaemia (pulpy kidney disease) by vaccination. Bull Int Office Epizootics 67: 1539±1567 8. Oxer D.T., Minty D.W. and Liefman C.E. (1971) Vaccination trials in sheep with clostridial vaccines with special reference to passively acquired Clostridium welchii type D antitoxin in lambs. Austral Vet J 47: 134±140 9. Jansen B.C. (1960) The Experimental reproduction of pulpy kidney disease. J South African Vet Med Assoc 31: 205±208 10. Shanks P.L. (1949) Enterotoxaemia in goats. Vet Record 61: 262±264 11. Uzal F.A., Nielsen K. and Kelly W.R. (1997) Competitive and indirect ELISA detection of Clostridium perfringens type D epsilon antitoxin in serum of goats. Vet Microbiol 24 51: 223±231 12. Moore D.S. and McCabe G.P. (1993) Introduction to the Practice of Statistics, 2nd ed. W.H. Freeman and Company, New York, USA. pp. 713±789 13. Uzal F.A. and Kelly W.R. (1998) Protection of goats against experimental enterotoxaemia by vaccination with Clostridium perfringens type D epsilon toxoid. Vet Record 24 142: 722±725 14. Smith L.D.S. and Marsh H. (1953) The immunization of young lambs against enterotoxaemia. Am J Vet Res 14: 408±410 15. Green D.S., Green M.J., Hillyer M.H. and Morgan K.L. (1987) Injection site reactions and antibody responses in sheep and goats after the use of a multivalent clostridial vaccine. Vet Record 120: 435±439