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Resistance to Taenia pisiformis larvea in rabbits: Immunisation using live organism followed by chemotherapy therapy
Veterinary Parasitology, 1 (1975) 159--163 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
RESISTANCE TO TAENIA PISIFORMIS LARVAE IN RABBITS: IMMUNISATION USING LIVE ORGANISMS FOLLOWED BY CHEMOTHERAPY
D.D. HEATH* and R.A.F. CHEVIS** *Department of Zoology, The Australian National University, Canberra, A. C. T. (Australia) **Ethnor Pry. Ltd, North Ryde, N.S.W. (Australia) *Present address: Wallaceville Animal Research Centre, Upper Hutt (New Zealand) (Received April 22nd, 1975)
ABSTRACT Heath, D.D. and Chevis, R.A.F., 1975. Resistance to Taenia pisiformis larvae in rabbits: Immunisation using live organisms followed by chemotherapy. Vet. Parasitol., 1: 159-163. Viable eggs or activated oncospheres of Taenia pisiformis were inoculated subcutaneously into rabbits. At various inte_~als the developing larvae were killed by treatment with Mebendazole. Most rabbits receiving oncospheres were protected against challenge infection if they were treated with Mebendazole 1 day after injection and absolute immunity was established in all rabbits if larvae were allowed to develop for 14 days before being killed. In rabbits receiving eggs, 21 days from injection to Mebendazole treatment was required before absolute immunity developed. Eggs appear to require a period of 1--2 weeks for hatching and oncosphere activation in a subcutaneous site. The data also indicate that production of functionally protective antigens occurs early during larval development. INTRODUCTIO~ G e m m e l l ( 1 9 6 5 ) and H e a t h ( 1 9 7 3 b a n d c) h a v e s h o w n t h a t in r a b b i t s a s t r o n g resistance t o i n f e c t i o n w i t h Taenia p i s i f o r m i s larvae is s t i m u l a t e d b y a p a r e n t e r a l i n o c u l a t i o n o f viable h o m o l o g o u s eggs or a c t i v a t e d o n c o s p h e r e s . Small h e p a t i c lesions w e r e c o m m o n l y o b s e r v e d f o l l o w i n g challenge with T. p i s i f o r m i s eggs, b u t t h e resistance t o successful d e v e l o p m e n t o f viable larvae was a b s o l u t e in m o s t rabbits. T h e f e w i m m u n i s e d r a b b i t s in w h i c h viable larvae d e v e l o p e d s h o w e d 9 7 - - 9 9 % p r o t e c t i o n . H o w e v e r , t h e d i s a d v a n t a g e o f this t y p e o f i m m u n i s a t i o n p r o c e d u r e was t h a t larvae o f t e n d e v e l o p e d t o m a t u r i t y a t t h e i n j e c t i o n site. M e b e n d a z o l e ( m e t h y l 5 - b e n z o y l b e n z i m i d a z o l e - 2 - c a r b a m a t e , J a n s s e n Pharm a c e u t i c a , Belgium) c a u s e d t h e d e a t h o f all juvenile o r m a t u r e T. p i s i f o r m i s larvae in r a b b i t s if a d m i n i s t e r e d orally in f e e d f o r 14 d a y s a t a r a t e o f 1 g M e b e n d a z o l e / k g f e e d ( H e a t h et al., 1 9 7 5 ) . Using this drug, it b e c o m e s possible t o o v e r c o m e t h e d i s a d v a n t a g e o f t h e a b o v e i m m u n i s i n g p r o c e d u r e , a n d
160 also to determine the development period required by subcutaneously inoculated eggs or oncospheres in order to stimulate immunity. The time necessary for orally administered mebendazole to kill developing larvae is not accurately known, but is apparently less than 14 days and may be even less than 7 days (Heath et al., 1975). A comparison between eggs and activated oncospheres was undertaken because activated oncospheres immediately begin larval development in a subcutaneous site (Heath, 1973 c), but eggs require time before activated oncospheres finally emerge. The process of activation in parenterally inoculated eggs has not been studied. The advantage of being able to use eggs to effect immunisation are numerous. No special procedures are required once eggs are sterilised (Heath, 1973 a), and cestode eggs have a long storage life. Heath (1970) f o u n d that 12% of T . p i s i f o r m i s eggs could still yield activated oncospheres after 294 days of storage at 4 ° C. MATERIALS AND METHODS - - Seventy-two 10-week-old New Zealand white rabbits free of cestode larvae were used. They were all maintained in one contaminationcontrolled room. They were divided into 12 groups, each of six rabbits. Each group containing three males and three females. Rabbit pellets (125 g/day) and water ad libitum were available to each individually-caged rabbit.
Rabbits.
Taenia pisiformis e g g s . - - Eggs were collected from worms from laboratoryreared dogs at necropsy. These eggs were stored sterile in phosphate-buffered saline solution (pH 7.2) following the technique of Heath (1973 a). Test activation of oncospheres (Heath and Smyth, 1970) showed that 70% of eggs were capable of yielding activated oncospheres. Suspensions used for inoculations therefore contained either 2000 eggs or 1400 activated oncospheres. - - Injections of eggs or oncospheres were made subcutaneously in the left mid-thoracic area. Five groups of six rabbits each received injections of eggs, and a further five groups received activated oncospheres. Care was taken that no contamination of the environment occurred which could result in accidental oral infection prior to the time of challenge infection. Twelve rabbits remained as uninjected controls.
Immunisation.
treatment. - - Mebendazole was incorporated into a pelleted ration similar to that which rabbits normally received. It was placed in the premix of vitamins and trace elements so that complete mixing was obtained. Rabbits received their standard dry matter aliquot of this ration for 14 days. New groups were offered the medicated feed at weekly intervals as shown in Table I. The ration contained 1 g Mebendazole/kg feed which was approximately 50 mg/kg body weight/day. All the medicated food was consumed by
Mebendazole
161
all rabbits, and n o side e f f e c t s were observed. M e b e n d a z o l e was also administered t o t h e u n i n o c u l a t e d animals, t o e x a m i n e t h e possibility o f Mebendazole residues having a p r o p h y l a c t i c e f f e c t o n challenge infections. Challenge i n f e c t i o n and n e c r o p s y . - - 4 days a f t e r t h e e n d o f t h e last series o f m e b e n d a z o l e feedings all rabbits were each a d m i n i s t e r e d 2 0 0 0 T. p i s i f o r m i s eggs orally. This m e a n t t h a t f o r all rabbits a t o t a l o f 39 days h a d elapsed bet w e e n i n o c u l a t i o n and challenge. T e s t activation at t h a t t i m e s h o w e d t h a t 50% o f eggs were capable o f yielding activated o n c o s p h e r e s . 28 t o 32 days a f t e r challenge, all rabbits were e x a m i n e d p o s t m o r t e m . A f t e r skinning, s u b c u t a n e o u s lesions were inspected. T h e inguinal canal and o m e n t u m w e r e t h e n s e a r c h e d f o r e n c y s t e d larvae t h a t m a y have r e s u l t e d f r o m previous accidental infections. T h e liver and intestines were t h e n rem o v e d and t h e p e r i t o n e a l cavity e x a m i n e d f o r larvae. T h e c o n d i t i o n o f t h e liver was r e c o r d e d , and t h e intestinal mass was rinsed in w a t e r so as t o wash o f f a n y u n e n c y s t e d i m m a t u r e cysticerci t h a t m a y have resulted f r o m the challenge i n f e c t i o n .
RESULTS T h e results are p r e s e n t e d in Table I. Variances are n o t p r e s e n t e d , b u t app r o x i m a t e d t h e m e a n s in all cases. S u b c u t a n e o u s i n o c u l a t i o n with e i t h e r eggs or o n c o s p h e r e s , and w i t h o u t m e b e n d a z o l e t r e a t m e n t , was t o t a l l y effective in p r e v e n t i n g t h e d e v e l o p m e n t o f migrating larvae f o l l o w i n g oral challenge (P < 0.001). H o w e v e r , s o m e live larvae were f o u n d in m o s t o f the i n o c u l a t i o n TABLE I Results of oral challenge with Taenia pisiformis eggs, following subcutaneous inoculation of rabbits with homologous eggs or oncospheres and treatment with Mebendazole. Inoculum
Days until Mebendazole treatment
Mean number of subcutaneous live larvae
Mean number of liver spots
Mean number of larvae in peritoneal cavity
Eggs
1 7 14 21 No treatment
0 0 0 0 4.7
9.2 14.7 11.7 6.8 5.5
18.7 9.0 8.1 0 0
Oncospheres
1 7 14 21 No treatment
0 0 0 0 4.2
14.5 8.8 8.8 3.2 0
1.5 2.0 0 0 0
Controls
21
0.7
51.3
162 sites. In those rabbits receiving the medicated feed, mebendazole was totally effective in killing all subcutaneous larvae that may have developed following inoculation. Activated oncospheres developing subcutaneously for 14 days or more appeared to stimulate a good resistance to development of migrating larvae (P < 0.001). However, as evidenced by the numbers of small liver lesions observed, the immunity was not as complete as in those animals where subcutaneous larvae had not been killed by mebendazole. Subcutaneous injection of eggs also stimulated good immunity, (P < 0.001) but the time required for this appeared to be 7--14 days more than for activated oncospheres. DISCUSSION Developing T. pisiformis larvae apparently only release antigens capable of stimulating absolute protective immunity for the first 15 days of larval development in vitro (Heath, 1973 b). Also, following subcutaneous inoculation of oncospheres, protective immunity seemed to increase gradually. The numbers of migrating larvae 9 days after immunisation were limited and the numbers of small liver lesions were progressively limited for 21 days, (Heath, 1973 c). Similar results were obtained in the present experiments, even though the immunising infections were killed by mebendazole. It is therefore probable that by 21 days of development from the oncosphere the total protective immunising potential of subcutaneous larvae had been realised. The results reported here concerning the immunogenicity of parenterally injected eggs, agree with those of Gemmell (1965). However, because T. pisiformis eggs injected subcutaneously appear to require 7--14 days before activated oncospheres are released, it is possible that enhanced immunity would be achieved if larvae were allowed to develop subcutaneously for 4 weeks before being killed with mebendazole. Until the immunising antigens of larval cestodes can be purified from in vitro culture and presented in a stable form, the method of immunisation presented here might be usefully applied to other larval cestodes in domestic livestock. Lot-fed cattle and pigs could receive the drug mixed in their ration without undue difficulty, but for free-ranging livestock the drug may require presentation as a slowly-released pellet. A further factor for consideration is that under field conditions, immunity is probably maintained by continuous oral challenge with eggs (Gemmell, 1972). The time for which the immunity stimulated in the present experiments will last in the absence of oral challenge, has yet to be determined. ACKNOWLEDGEMENT The work reported here was financed by a grant from the Australian Meat Research Committee.
163
REFERENCES Gemmell, M.A., 1965. Immunological responses of the mammalian host against tapeworm infections. II. Species specificity of hexacanth embryos in protecting rabbits against Taenia pisiformis. Immunology, 8: 270--280. Gemmell, M.A., 1972. Hydatidosis and cysticercosis. 5. Some problems of inducing resistance to Taenia hydatigena under conditions of a strong infection pressure. Aust. Vet. J., 48: 29--31. Heath, D.D., 1970. The developmental biology of larval cyclophyllidean cestodes in mammals. Thesis, Aust. Natl Univ., Canberra, A.C.T., 235 pp. Heath, D.D., 1973 a. An improved technique for the in vitro culture of taeniid larvae. Int. J. Parasitol., 3: 481--484. Heath, D.D., 1973 b. Resistance to Taenia pisiformis larvae in rabbits. I. Examination of the antigenically protective phase of larval development. Int. J. Parasitol., 3: 485--489. Heath, D.D., 1~73 c. Resistance to Taenia pisiformis larvae in rabbits. II. Temporal relationships and the development phase affected. Int. J. Parasitol., 3: 491--497. Heath, D.D. and Smyth, J.D., 1970. In vitro cultivation of Echinococcus granulosus, Taenia hydatigena, T. ovis, T. pisiformis and T. serialis from oncosphere to cystic larva. Parasitology, 61: 329--343. Heath, D.D., Christie, M.J. and Chevis, R.A.F., 1975. The lethal effect of mebendazole on secondary Echinococcus granulosus, cysticerci of Taenia pisiformis and tetrathyridia of Mesocestoides corti. Parasitology, 70: 273--285.
RESISTANCE TO TAENIA PISIFORMIS LARVAE IN RABBITS: IMMUNISATION USING LIVE ORGANISMS FOLLOWED BY CHEMOTHERAPY
D.D. HEATH* and R.A.F. CHEVIS** *Department of Zoology, The Australian National University, Canberra, A. C. T. (Australia) **Ethnor Pry. Ltd, North Ryde, N.S.W. (Australia) *Present address: Wallaceville Animal Research Centre, Upper Hutt (New Zealand) (Received April 22nd, 1975)
ABSTRACT Heath, D.D. and Chevis, R.A.F., 1975. Resistance to Taenia pisiformis larvae in rabbits: Immunisation using live organisms followed by chemotherapy. Vet. Parasitol., 1: 159-163. Viable eggs or activated oncospheres of Taenia pisiformis were inoculated subcutaneously into rabbits. At various inte_~als the developing larvae were killed by treatment with Mebendazole. Most rabbits receiving oncospheres were protected against challenge infection if they were treated with Mebendazole 1 day after injection and absolute immunity was established in all rabbits if larvae were allowed to develop for 14 days before being killed. In rabbits receiving eggs, 21 days from injection to Mebendazole treatment was required before absolute immunity developed. Eggs appear to require a period of 1--2 weeks for hatching and oncosphere activation in a subcutaneous site. The data also indicate that production of functionally protective antigens occurs early during larval development. INTRODUCTIO~ G e m m e l l ( 1 9 6 5 ) and H e a t h ( 1 9 7 3 b a n d c) h a v e s h o w n t h a t in r a b b i t s a s t r o n g resistance t o i n f e c t i o n w i t h Taenia p i s i f o r m i s larvae is s t i m u l a t e d b y a p a r e n t e r a l i n o c u l a t i o n o f viable h o m o l o g o u s eggs or a c t i v a t e d o n c o s p h e r e s . Small h e p a t i c lesions w e r e c o m m o n l y o b s e r v e d f o l l o w i n g challenge with T. p i s i f o r m i s eggs, b u t t h e resistance t o successful d e v e l o p m e n t o f viable larvae was a b s o l u t e in m o s t rabbits. T h e f e w i m m u n i s e d r a b b i t s in w h i c h viable larvae d e v e l o p e d s h o w e d 9 7 - - 9 9 % p r o t e c t i o n . H o w e v e r , t h e d i s a d v a n t a g e o f this t y p e o f i m m u n i s a t i o n p r o c e d u r e was t h a t larvae o f t e n d e v e l o p e d t o m a t u r i t y a t t h e i n j e c t i o n site. M e b e n d a z o l e ( m e t h y l 5 - b e n z o y l b e n z i m i d a z o l e - 2 - c a r b a m a t e , J a n s s e n Pharm a c e u t i c a , Belgium) c a u s e d t h e d e a t h o f all juvenile o r m a t u r e T. p i s i f o r m i s larvae in r a b b i t s if a d m i n i s t e r e d orally in f e e d f o r 14 d a y s a t a r a t e o f 1 g M e b e n d a z o l e / k g f e e d ( H e a t h et al., 1 9 7 5 ) . Using this drug, it b e c o m e s possible t o o v e r c o m e t h e d i s a d v a n t a g e o f t h e a b o v e i m m u n i s i n g p r o c e d u r e , a n d
160 also to determine the development period required by subcutaneously inoculated eggs or oncospheres in order to stimulate immunity. The time necessary for orally administered mebendazole to kill developing larvae is not accurately known, but is apparently less than 14 days and may be even less than 7 days (Heath et al., 1975). A comparison between eggs and activated oncospheres was undertaken because activated oncospheres immediately begin larval development in a subcutaneous site (Heath, 1973 c), but eggs require time before activated oncospheres finally emerge. The process of activation in parenterally inoculated eggs has not been studied. The advantage of being able to use eggs to effect immunisation are numerous. No special procedures are required once eggs are sterilised (Heath, 1973 a), and cestode eggs have a long storage life. Heath (1970) f o u n d that 12% of T . p i s i f o r m i s eggs could still yield activated oncospheres after 294 days of storage at 4 ° C. MATERIALS AND METHODS - - Seventy-two 10-week-old New Zealand white rabbits free of cestode larvae were used. They were all maintained in one contaminationcontrolled room. They were divided into 12 groups, each of six rabbits. Each group containing three males and three females. Rabbit pellets (125 g/day) and water ad libitum were available to each individually-caged rabbit.
Rabbits.
Taenia pisiformis e g g s . - - Eggs were collected from worms from laboratoryreared dogs at necropsy. These eggs were stored sterile in phosphate-buffered saline solution (pH 7.2) following the technique of Heath (1973 a). Test activation of oncospheres (Heath and Smyth, 1970) showed that 70% of eggs were capable of yielding activated oncospheres. Suspensions used for inoculations therefore contained either 2000 eggs or 1400 activated oncospheres. - - Injections of eggs or oncospheres were made subcutaneously in the left mid-thoracic area. Five groups of six rabbits each received injections of eggs, and a further five groups received activated oncospheres. Care was taken that no contamination of the environment occurred which could result in accidental oral infection prior to the time of challenge infection. Twelve rabbits remained as uninjected controls.
Immunisation.
treatment. - - Mebendazole was incorporated into a pelleted ration similar to that which rabbits normally received. It was placed in the premix of vitamins and trace elements so that complete mixing was obtained. Rabbits received their standard dry matter aliquot of this ration for 14 days. New groups were offered the medicated feed at weekly intervals as shown in Table I. The ration contained 1 g Mebendazole/kg feed which was approximately 50 mg/kg body weight/day. All the medicated food was consumed by
Mebendazole
161
all rabbits, and n o side e f f e c t s were observed. M e b e n d a z o l e was also administered t o t h e u n i n o c u l a t e d animals, t o e x a m i n e t h e possibility o f Mebendazole residues having a p r o p h y l a c t i c e f f e c t o n challenge infections. Challenge i n f e c t i o n and n e c r o p s y . - - 4 days a f t e r t h e e n d o f t h e last series o f m e b e n d a z o l e feedings all rabbits were each a d m i n i s t e r e d 2 0 0 0 T. p i s i f o r m i s eggs orally. This m e a n t t h a t f o r all rabbits a t o t a l o f 39 days h a d elapsed bet w e e n i n o c u l a t i o n and challenge. T e s t activation at t h a t t i m e s h o w e d t h a t 50% o f eggs were capable o f yielding activated o n c o s p h e r e s . 28 t o 32 days a f t e r challenge, all rabbits were e x a m i n e d p o s t m o r t e m . A f t e r skinning, s u b c u t a n e o u s lesions were inspected. T h e inguinal canal and o m e n t u m w e r e t h e n s e a r c h e d f o r e n c y s t e d larvae t h a t m a y have r e s u l t e d f r o m previous accidental infections. T h e liver and intestines were t h e n rem o v e d and t h e p e r i t o n e a l cavity e x a m i n e d f o r larvae. T h e c o n d i t i o n o f t h e liver was r e c o r d e d , and t h e intestinal mass was rinsed in w a t e r so as t o wash o f f a n y u n e n c y s t e d i m m a t u r e cysticerci t h a t m a y have resulted f r o m the challenge i n f e c t i o n .
RESULTS T h e results are p r e s e n t e d in Table I. Variances are n o t p r e s e n t e d , b u t app r o x i m a t e d t h e m e a n s in all cases. S u b c u t a n e o u s i n o c u l a t i o n with e i t h e r eggs or o n c o s p h e r e s , and w i t h o u t m e b e n d a z o l e t r e a t m e n t , was t o t a l l y effective in p r e v e n t i n g t h e d e v e l o p m e n t o f migrating larvae f o l l o w i n g oral challenge (P < 0.001). H o w e v e r , s o m e live larvae were f o u n d in m o s t o f the i n o c u l a t i o n TABLE I Results of oral challenge with Taenia pisiformis eggs, following subcutaneous inoculation of rabbits with homologous eggs or oncospheres and treatment with Mebendazole. Inoculum
Days until Mebendazole treatment
Mean number of subcutaneous live larvae
Mean number of liver spots
Mean number of larvae in peritoneal cavity
Eggs
1 7 14 21 No treatment
0 0 0 0 4.7
9.2 14.7 11.7 6.8 5.5
18.7 9.0 8.1 0 0
Oncospheres
1 7 14 21 No treatment
0 0 0 0 4.2
14.5 8.8 8.8 3.2 0
1.5 2.0 0 0 0
Controls
21
0.7
51.3
162 sites. In those rabbits receiving the medicated feed, mebendazole was totally effective in killing all subcutaneous larvae that may have developed following inoculation. Activated oncospheres developing subcutaneously for 14 days or more appeared to stimulate a good resistance to development of migrating larvae (P < 0.001). However, as evidenced by the numbers of small liver lesions observed, the immunity was not as complete as in those animals where subcutaneous larvae had not been killed by mebendazole. Subcutaneous injection of eggs also stimulated good immunity, (P < 0.001) but the time required for this appeared to be 7--14 days more than for activated oncospheres. DISCUSSION Developing T. pisiformis larvae apparently only release antigens capable of stimulating absolute protective immunity for the first 15 days of larval development in vitro (Heath, 1973 b). Also, following subcutaneous inoculation of oncospheres, protective immunity seemed to increase gradually. The numbers of migrating larvae 9 days after immunisation were limited and the numbers of small liver lesions were progressively limited for 21 days, (Heath, 1973 c). Similar results were obtained in the present experiments, even though the immunising infections were killed by mebendazole. It is therefore probable that by 21 days of development from the oncosphere the total protective immunising potential of subcutaneous larvae had been realised. The results reported here concerning the immunogenicity of parenterally injected eggs, agree with those of Gemmell (1965). However, because T. pisiformis eggs injected subcutaneously appear to require 7--14 days before activated oncospheres are released, it is possible that enhanced immunity would be achieved if larvae were allowed to develop subcutaneously for 4 weeks before being killed with mebendazole. Until the immunising antigens of larval cestodes can be purified from in vitro culture and presented in a stable form, the method of immunisation presented here might be usefully applied to other larval cestodes in domestic livestock. Lot-fed cattle and pigs could receive the drug mixed in their ration without undue difficulty, but for free-ranging livestock the drug may require presentation as a slowly-released pellet. A further factor for consideration is that under field conditions, immunity is probably maintained by continuous oral challenge with eggs (Gemmell, 1972). The time for which the immunity stimulated in the present experiments will last in the absence of oral challenge, has yet to be determined. ACKNOWLEDGEMENT The work reported here was financed by a grant from the Australian Meat Research Committee.
163
REFERENCES Gemmell, M.A., 1965. Immunological responses of the mammalian host against tapeworm infections. II. Species specificity of hexacanth embryos in protecting rabbits against Taenia pisiformis. Immunology, 8: 270--280. Gemmell, M.A., 1972. Hydatidosis and cysticercosis. 5. Some problems of inducing resistance to Taenia hydatigena under conditions of a strong infection pressure. Aust. Vet. J., 48: 29--31. Heath, D.D., 1970. The developmental biology of larval cyclophyllidean cestodes in mammals. Thesis, Aust. Natl Univ., Canberra, A.C.T., 235 pp. Heath, D.D., 1973 a. An improved technique for the in vitro culture of taeniid larvae. Int. J. Parasitol., 3: 481--484. Heath, D.D., 1973 b. Resistance to Taenia pisiformis larvae in rabbits. I. Examination of the antigenically protective phase of larval development. Int. J. Parasitol., 3: 485--489. Heath, D.D., 1~73 c. Resistance to Taenia pisiformis larvae in rabbits. II. Temporal relationships and the development phase affected. Int. J. Parasitol., 3: 491--497. Heath, D.D. and Smyth, J.D., 1970. In vitro cultivation of Echinococcus granulosus, Taenia hydatigena, T. ovis, T. pisiformis and T. serialis from oncosphere to cystic larva. Parasitology, 61: 329--343. Heath, D.D., Christie, M.J. and Chevis, R.A.F., 1975. The lethal effect of mebendazole on secondary Echinococcus granulosus, cysticerci of Taenia pisiformis and tetrathyridia of Mesocestoides corti. Parasitology, 70: 273--285.