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Echinococcus granulosus: Ovicidal activity of praziquantel and bunamidine hydrochloride
EXPERIMENTAL
PARASITOLOGY
Echinococcus
47,
131-133
Pan American
Ovicidol Activity Bunamidine Hydrochloride
of Praziquantel
granulosus:
and AMAR
(1979)
S. THAKUR, Zoonoses
U. PREZIOSO, AND N. MARCHEVSKY
Center, PAHOIWHO, 1000 Buenos Aires,
(Accepted
for
publication
Casilla Argentina 5 May
309%CoTTeo
Central,
1978)
THAKUR, A. S., PREZIOSO, U., AND MARCHEVSKY, N. 1979. Echinococcus granulosus: ovicidal activity of praziquantel and bunamidine hydrochloride. Experimental Parasitology 47, 131-133. Eggs of Echinococcus granulosus either released from or contained within the proglottids were treated with praziquantel and bunamidine hydrochloride at 37 C for 1 hr and then administered orally to mice and gerbils to test their infectivity. Praziquantel did not possess absolute ovicidal activity against E. granulosus eggs either within or outside the proglottids and bunamidine hydrochloride did not kill them within the proglottids. These findings bear on the use of praziquantel in control programs. INDEX DESCRIPTORS: Echinococcus granulosus; Cestode; Praziquantel; Bunamidine hydrochloride; Ovicide; Hydatid disease, control; Mouse; Gerbil.
Praziquantel (Droncit, Bayer) has been found 100% effective against adult Echinococcus granulosus in dogs (Thakur et al. 1978) and several Latin American countries have planned to use it in their hydatid disease control programs. Bunamidine hydrochloride inhibits infectivity of E. granulosus eggs outside the proglottids when they are incubated with the drug at 37 C for 2 hr (Williams et al. 1973) but no information is available on its effects on the eggs within the proglottids. Because both these drugs are used in the treatment of dogs, studies of their effects on the infectivity of E. granulosus eggs either released from or contained within the proglottids would be most useful. MATERIALS
AND
METHODS
EcMnococcus granulosus hydatid cysts were obtained from sheep slaughtered at
abbatoirs in the province of Buenos Aires, Argentina. An amount of 0.5 ml of freshly packed protoscolices from several pool cysts was mixed with minced meat and fed to each of several dogs of different age, breed, and sex. Food was withdrawn from the dogs 24 hr before they were killed, 14 weeks postinfection. Intestines were cut open and kept in saline at 4 C. Gravid proglottids were collected and stored at refrigerator temperature. Eggs were removed by microdissection to obtain a clean suspension. The eggs were stored at 4 C in sterile saline with 1000 IU penicillin/ml and 1 mg/ml streptomycin and were used for studies within 4 days after they had been collected. Released eggs as we11 as those contained within the proglottids were incubated with different dilutions of praziquantel and bunamidine hydrochIoride at 37 C (Table I), along with the controls, in a water bath with shaker for 1 hr.
131 0014-4894/79/020131-03$02.00/O All
Copyright @ 1979 rights d reproduction
by Academic Press, Inc. in any form reserved.
132
THAKWR,
PREZIOSO,
AND
TABLE Infectivity Experimental
animals
of Echinococcus and type
granulosus
of treatment
1. Mice A. Eggs contained within the proglottids (1) Control (HZO) (2) Praziquantel 1 mg/ml (HzO) (3) Praziquantel 0.1 mg/ml (H20) (4) Control (HZO) + cremophore (5) Praziquantel 1 mg/ml (H,O) + cremophore (6) Praziquantel 0.1 mg/ml (HZO) + cremophore (7) Bunamidine hydrochloride 11.1 mg/ml (HZO) B. Eggs (8) (9) (10) (11) (12) (13) II.
released from proglottids Control (HgO) Praziquantel 1 mg/ml (HZO) PraziquantelO.1 mg/ml (HZO) Control (H,O) + cremophore Praziquantel 1 mg/ml (H,O) + cremophore Praziquantel 0.1 mg/ml (H20) + cremophore
Gerbils A. Eggs released from proglottids (14) Control (HzO) + cremophore (15) Praziquantel 1 mg/ml (H,O)
+ cremophore
Praziquantel is insoluble in water and in order to make a uniform suspension of the drug, cremophore (BASF, Germany) was added at a ratio of 0.2 ml/100 mg praziquantel to another group of tubes with different dilutions of the drug as well as to the controls at a rate of 0.2 ml/100 ml of distilled water. After treatment with the drugs, the eggs contained within the proglottids were released by microdissection and completely freed from the remainders of the proglottids. Both the control and drug-treated eggs of all the groups were washed in sterile saline and mature eggs were counted and were orally administered in the amount of 1000 to 21-day-old mice CF1 strain and gerbils, which were killed four months later to examine the development of cysts. RESULTS
The cours_es of development of Echinococcus granulosus cysts in mice and gerbils
MARCHEVSKY
I Eggs
in CFi
No. of animals inoculated
Mice
and Gerbils
No. of animals surviving 4 months later
No. of animals found infected
Percentage of eggs which developed into cysts
17 19 20 11 1.5 18 17
10 15 17 6 13 13 12
10 15 12 6 13 12 12
1.86 1.74 1.62 1.76 1.45 1.6<5 1.70
20 20 20 20 20 18
12 19 15 17 18 14
11 14 13 17 17 13
1.77 0.71 0.73 1.1 0.54 0.85
10 20
9 17
9 13
0.36 0.33
inoculated with control and drug-treated eggs are described in Table I. Although the percentage of eggs established in mice was lower when the eggs had been treated with praziquantel as compared to the controls, the drug did not have absolute ovitidal effect. The percentage of eggs established in mice was greater when the treated eggs were contained within the proglottids as compared to released eggs. Bunamidine hydrochloride did not kill the eggs contained within the proglottids. DISCUSSION
The eggs of E. granulosus are very resistant; they were still infective for sheep after 4 winter months on grass plots in New Zealand (Sweatman and Williams 1963) and survived at -30 C with no detectable effect on infectivity (Colli and Williams 1972). Nosik ( 1952) reported that eggs of E. granulosus were resistant to formol, and
Echinococcus
grandosus:
Parnell (1965) pointed out that unlike other helminths which depend on number to produce disease, hydatid disease may develop if only one embryo becomes established, therefore any substance which does not have absolute ovicidal activity cannot be considered safe. Bunamidine hydrochloride kills eggs that have been released from proglottids after treatment for 2 hr at 37 C (Williams et al. 1973). In the present study the drug failed to kill eggs contained within proglottids. The morphological changes resulting in the shrinking of embryo and oncospheral membrane from the embryophore seen in the bunamidine treated eggs of Taenia taeniaeformis, T. pisiformis, and T. saginata (Williams et al. 1973) were not noticed in bunamidine-treated eggs of E. granulosus. A drug which is not only a taenicide but also ovicidal would be of great value in hydatid disease control programs. Although praziquantel is 100% effective against immature and gravid E. granulosus and is the drug of choice in the treatment of dogs (Thakur et al. 1978), the present studies have shown that it does not possess absolute ovicidal activity against E. granulosus eggs released from the proglottids and is ineffective against eggs contained within the proglottids or otherwise protected by organic material. The praziquantel-treated dog, if infected with gravid parasites, may liberate viable eggs on the day following the treatment and contaminate the environment. This limits the use of the drug in control programs for mass treatment. Dogs should therefore be kept in confinement for 1 day after the treatment, and afterward should be dosed within the prepatent period of the parasite, particularly when there is danger of reinfection through infected viscera. The percentage of control eggs which developed in mice after oral infection varied from 1.77 to 1.86 and is greater than the figure (0.438-0.470) obtained by P&ezEsandi et al. (1974). This may be attributable to the fact that in the present studies
133
OVICIDES
the eggs were not treated with the chemicals for their removal from the remainder of the proglottids and were used within 4 days after collection. A higher percentage of eggs developed in mice than in gerbils, thus confirming the greater susceptibility of mice to infection by E. granulosus eggs (Williams and Colli 1970). Since praziquantel is not ovicidal against E. granulosus eggs under in vitro conditions, further studies to define the ovicidal activity of the drug under in viva conditions, as well as the number, distribution, and viability of eggs from praziquanteltreated dogs are in progress in our laboratory. Proper preca&ois should be taken to prevent the contamination of the environment after treatment of the dogs infected with gravid parasites. REFERENCES COLLI, ence
C. W., AND WILLAMS, J. F. 1972. Influof temperature on the infectivity of eggs of Echinococcus granulosus in laboratory rodents. Journal of Parasitology 58, 422-426. NOSIK, A. F. 1952. Resistance of onchospheres of Echinococcus granulosus to influence of some physical and chemical factors. Proceedings Kharkov Veterinary Institute 21, 304-311. PARNELL, I. W. 1965. Some observations on taeniic ovicides: Screening techniques and the effects of some inorganic compounds. Journal of Helminthology 39, 257-272. PEREZ-ESANDI, M. V., COLLI, C. W., AND SCHANIZ, P. M. 1974. The ovicidal effect of selected chemicals against eggs of Echinococcus granulosus. Bulletin of the World Health OTganization 51, 550-551. SWEATMAN, G. K., AND WILLIAMS, R. J. 1963. Survival of Echinococcus granulosus and Taenfa hydatigena eggs in two extreme climatic regions of New Zealand. Research in Veterinary Science 4, 199-216. THAKUR, A. S., PREZIOSO, U., AND MARCHEVSKY, N. 1978. Efficacy of Droncit against Echinococcus granulosus infection in dogs. American Journal of Veterinary Research 39, 859-860. WILLIAMS, J. F., AND COLLI, C. W. 1970. Primary cystic infection with Echinococcus granuZosus and Taenia hydatigena in Meriones unguiculatus. Journal of Parasitology 56, 509-513. WILLIAMS, J. F. COLLI, C. W., LEID, R. W., AND MACARTHUR, R. 1973. The effect of bunamidine hydrochloride on the infectivity of taeniid ova. Journal of Parasitology 59, 1141-1144.
PARASITOLOGY
Echinococcus
47,
131-133
Pan American
Ovicidol Activity Bunamidine Hydrochloride
of Praziquantel
granulosus:
and AMAR
(1979)
S. THAKUR, Zoonoses
U. PREZIOSO, AND N. MARCHEVSKY
Center, PAHOIWHO, 1000 Buenos Aires,
(Accepted
for
publication
Casilla Argentina 5 May
309%CoTTeo
Central,
1978)
THAKUR, A. S., PREZIOSO, U., AND MARCHEVSKY, N. 1979. Echinococcus granulosus: ovicidal activity of praziquantel and bunamidine hydrochloride. Experimental Parasitology 47, 131-133. Eggs of Echinococcus granulosus either released from or contained within the proglottids were treated with praziquantel and bunamidine hydrochloride at 37 C for 1 hr and then administered orally to mice and gerbils to test their infectivity. Praziquantel did not possess absolute ovicidal activity against E. granulosus eggs either within or outside the proglottids and bunamidine hydrochloride did not kill them within the proglottids. These findings bear on the use of praziquantel in control programs. INDEX DESCRIPTORS: Echinococcus granulosus; Cestode; Praziquantel; Bunamidine hydrochloride; Ovicide; Hydatid disease, control; Mouse; Gerbil.
Praziquantel (Droncit, Bayer) has been found 100% effective against adult Echinococcus granulosus in dogs (Thakur et al. 1978) and several Latin American countries have planned to use it in their hydatid disease control programs. Bunamidine hydrochloride inhibits infectivity of E. granulosus eggs outside the proglottids when they are incubated with the drug at 37 C for 2 hr (Williams et al. 1973) but no information is available on its effects on the eggs within the proglottids. Because both these drugs are used in the treatment of dogs, studies of their effects on the infectivity of E. granulosus eggs either released from or contained within the proglottids would be most useful. MATERIALS
AND
METHODS
EcMnococcus granulosus hydatid cysts were obtained from sheep slaughtered at
abbatoirs in the province of Buenos Aires, Argentina. An amount of 0.5 ml of freshly packed protoscolices from several pool cysts was mixed with minced meat and fed to each of several dogs of different age, breed, and sex. Food was withdrawn from the dogs 24 hr before they were killed, 14 weeks postinfection. Intestines were cut open and kept in saline at 4 C. Gravid proglottids were collected and stored at refrigerator temperature. Eggs were removed by microdissection to obtain a clean suspension. The eggs were stored at 4 C in sterile saline with 1000 IU penicillin/ml and 1 mg/ml streptomycin and were used for studies within 4 days after they had been collected. Released eggs as we11 as those contained within the proglottids were incubated with different dilutions of praziquantel and bunamidine hydrochIoride at 37 C (Table I), along with the controls, in a water bath with shaker for 1 hr.
131 0014-4894/79/020131-03$02.00/O All
Copyright @ 1979 rights d reproduction
by Academic Press, Inc. in any form reserved.
132
THAKWR,
PREZIOSO,
AND
TABLE Infectivity Experimental
animals
of Echinococcus and type
granulosus
of treatment
1. Mice A. Eggs contained within the proglottids (1) Control (HZO) (2) Praziquantel 1 mg/ml (HzO) (3) Praziquantel 0.1 mg/ml (H20) (4) Control (HZO) + cremophore (5) Praziquantel 1 mg/ml (H,O) + cremophore (6) Praziquantel 0.1 mg/ml (HZO) + cremophore (7) Bunamidine hydrochloride 11.1 mg/ml (HZO) B. Eggs (8) (9) (10) (11) (12) (13) II.
released from proglottids Control (HgO) Praziquantel 1 mg/ml (HZO) PraziquantelO.1 mg/ml (HZO) Control (H,O) + cremophore Praziquantel 1 mg/ml (H,O) + cremophore Praziquantel 0.1 mg/ml (H20) + cremophore
Gerbils A. Eggs released from proglottids (14) Control (HzO) + cremophore (15) Praziquantel 1 mg/ml (H,O)
+ cremophore
Praziquantel is insoluble in water and in order to make a uniform suspension of the drug, cremophore (BASF, Germany) was added at a ratio of 0.2 ml/100 mg praziquantel to another group of tubes with different dilutions of the drug as well as to the controls at a rate of 0.2 ml/100 ml of distilled water. After treatment with the drugs, the eggs contained within the proglottids were released by microdissection and completely freed from the remainders of the proglottids. Both the control and drug-treated eggs of all the groups were washed in sterile saline and mature eggs were counted and were orally administered in the amount of 1000 to 21-day-old mice CF1 strain and gerbils, which were killed four months later to examine the development of cysts. RESULTS
The cours_es of development of Echinococcus granulosus cysts in mice and gerbils
MARCHEVSKY
I Eggs
in CFi
No. of animals inoculated
Mice
and Gerbils
No. of animals surviving 4 months later
No. of animals found infected
Percentage of eggs which developed into cysts
17 19 20 11 1.5 18 17
10 15 17 6 13 13 12
10 15 12 6 13 12 12
1.86 1.74 1.62 1.76 1.45 1.6<5 1.70
20 20 20 20 20 18
12 19 15 17 18 14
11 14 13 17 17 13
1.77 0.71 0.73 1.1 0.54 0.85
10 20
9 17
9 13
0.36 0.33
inoculated with control and drug-treated eggs are described in Table I. Although the percentage of eggs established in mice was lower when the eggs had been treated with praziquantel as compared to the controls, the drug did not have absolute ovitidal effect. The percentage of eggs established in mice was greater when the treated eggs were contained within the proglottids as compared to released eggs. Bunamidine hydrochloride did not kill the eggs contained within the proglottids. DISCUSSION
The eggs of E. granulosus are very resistant; they were still infective for sheep after 4 winter months on grass plots in New Zealand (Sweatman and Williams 1963) and survived at -30 C with no detectable effect on infectivity (Colli and Williams 1972). Nosik ( 1952) reported that eggs of E. granulosus were resistant to formol, and
Echinococcus
grandosus:
Parnell (1965) pointed out that unlike other helminths which depend on number to produce disease, hydatid disease may develop if only one embryo becomes established, therefore any substance which does not have absolute ovicidal activity cannot be considered safe. Bunamidine hydrochloride kills eggs that have been released from proglottids after treatment for 2 hr at 37 C (Williams et al. 1973). In the present study the drug failed to kill eggs contained within proglottids. The morphological changes resulting in the shrinking of embryo and oncospheral membrane from the embryophore seen in the bunamidine treated eggs of Taenia taeniaeformis, T. pisiformis, and T. saginata (Williams et al. 1973) were not noticed in bunamidine-treated eggs of E. granulosus. A drug which is not only a taenicide but also ovicidal would be of great value in hydatid disease control programs. Although praziquantel is 100% effective against immature and gravid E. granulosus and is the drug of choice in the treatment of dogs (Thakur et al. 1978), the present studies have shown that it does not possess absolute ovicidal activity against E. granulosus eggs released from the proglottids and is ineffective against eggs contained within the proglottids or otherwise protected by organic material. The praziquantel-treated dog, if infected with gravid parasites, may liberate viable eggs on the day following the treatment and contaminate the environment. This limits the use of the drug in control programs for mass treatment. Dogs should therefore be kept in confinement for 1 day after the treatment, and afterward should be dosed within the prepatent period of the parasite, particularly when there is danger of reinfection through infected viscera. The percentage of control eggs which developed in mice after oral infection varied from 1.77 to 1.86 and is greater than the figure (0.438-0.470) obtained by P&ezEsandi et al. (1974). This may be attributable to the fact that in the present studies
133
OVICIDES
the eggs were not treated with the chemicals for their removal from the remainder of the proglottids and were used within 4 days after collection. A higher percentage of eggs developed in mice than in gerbils, thus confirming the greater susceptibility of mice to infection by E. granulosus eggs (Williams and Colli 1970). Since praziquantel is not ovicidal against E. granulosus eggs under in vitro conditions, further studies to define the ovicidal activity of the drug under in viva conditions, as well as the number, distribution, and viability of eggs from praziquanteltreated dogs are in progress in our laboratory. Proper preca&ois should be taken to prevent the contamination of the environment after treatment of the dogs infected with gravid parasites. REFERENCES COLLI, ence
C. W., AND WILLAMS, J. F. 1972. Influof temperature on the infectivity of eggs of Echinococcus granulosus in laboratory rodents. Journal of Parasitology 58, 422-426. NOSIK, A. F. 1952. Resistance of onchospheres of Echinococcus granulosus to influence of some physical and chemical factors. Proceedings Kharkov Veterinary Institute 21, 304-311. PARNELL, I. W. 1965. Some observations on taeniic ovicides: Screening techniques and the effects of some inorganic compounds. Journal of Helminthology 39, 257-272. PEREZ-ESANDI, M. V., COLLI, C. W., AND SCHANIZ, P. M. 1974. The ovicidal effect of selected chemicals against eggs of Echinococcus granulosus. Bulletin of the World Health OTganization 51, 550-551. SWEATMAN, G. K., AND WILLIAMS, R. J. 1963. Survival of Echinococcus granulosus and Taenfa hydatigena eggs in two extreme climatic regions of New Zealand. Research in Veterinary Science 4, 199-216. THAKUR, A. S., PREZIOSO, U., AND MARCHEVSKY, N. 1978. Efficacy of Droncit against Echinococcus granulosus infection in dogs. American Journal of Veterinary Research 39, 859-860. WILLIAMS, J. F., AND COLLI, C. W. 1970. Primary cystic infection with Echinococcus granuZosus and Taenia hydatigena in Meriones unguiculatus. Journal of Parasitology 56, 509-513. WILLIAMS, J. F. COLLI, C. W., LEID, R. W., AND MACARTHUR, R. 1973. The effect of bunamidine hydrochloride on the infectivity of taeniid ova. Journal of Parasitology 59, 1141-1144.