EFFECTS OF MEBENDAZOLE AND LEVAMISOLE ON TETRATHYRIDIA OF MESOCESTOZDES CORTZ IN THE MOUSE EVA-MARIA BENNET, CAROLYN BEHM and C. BRYANT Department
of Zoology, Australian National University, Canberra, Australia (Received 20 December
1977)
Abstract-BENNET E.-M., BEHM C. A. and BRYANTC. 1978. Effects of mebendazole and levamisole on tetrathyridia of Mesocestoides corti in the mouse. International Journal for Parasitology 8: 463-466.
Mebendazole, but not levamisole, administered to mice carrying artificial infections of 50 tetrathyridia of Mesocestoides corti, was effective in killing the parasites. However, simultaneous administration of mebendazole and levamisole was still more effective. Treatment with levamisole before infection had no additional effect. Injection of mice with dead larvae offered some protection against a subsequent challenge with 50 live larvae; however, levamisole did not then improve the anthelmintic efficacy of mebendazole. In mice rendered immunoincompetent by radiation mebendazole was less effective than in nonirradiated controls and levamisole again did not enhance the effect of mebendazole. It is concluded that anthelmintic efficacy of mebendazole depends on its anthelmintic activity supplemented by the host’s immune response; and that levamisole stimulates the latter. 1NDEX KEY WORDS: anthelmintics;
mebendazole;
INTRODUCTION
SPECHT & VOGE (1965) found that tetrathyridia of Mesocestoides corti (Cestoda: Cyclophyllidea) multiply asexually in the liver and body cavity of mice
and can be serially transferred by intraperitoneal inoculation. Destruction of the liver by the larvae initially causeq a non-specific inflammatory response. Later, specific resistance to the parasite develops, which limits parasitic multiplication but does not prevent the eventual death of the mouse (Specht & Widmer, 1972). Immunity to tetrathyridia is both cellular and humoral (Pollacco, Nicholas, Mitchell and Stewart, 1978, submitted for publication). Mebendazole is a broad-spectrum anthelmintic, effective against adult cestodes and also capable of killing larva1 cestodes, including M. corti (Heath, Christie & Chevis, 1975). Levamisole is effective against nematodes at low concentrations (Van den Bossche & Janssen, 1969) but appears to be without action against cestodes. Its primary effect seems to be exerted on the nematode nervous system (Coles, 1974). Levamisole has also attracted attention because it stimulates the immune response (Renoux & Renoux, 1974). How it acts as an immunoadjuvant is unknown. In mice it is likely that the macrophage system is involved (Hoebke & Franchi, 1973). This paper describes the effect of levamisole in enhancing the anthelmintic activity of mebendazole against tetrathyridia of M. corti in mice. MATERIALS
AND
METHODS
A strain of M. corti, originally isolated by Specht &
levamisole; Mesocestoides corti.
Voge (I 965), was maintained by serial passage in a closed colony of outbred mice (Piebald strain). Only female mice were used in experiments: their ages ranged from 5 to 8 weeks at infection. Mebendazole was administered as a 1 g/kg constituent of a peanut meal formulated by Ethnor Pty Ltd, which formed the sole diet of the mice for the duration of the experiment. Such administration approximated a dose of 90-250 mg/kg body weight/day and falls well below the LDSOfor mice, which exceeds I g/kg body weight (Marsboom, 1973). The amount of mebendazole consumed by the mice was determined daily by weighing the medicated feed. Levamisole was administered in drinking water at a level which approximated a dose of IO mg/kg body weight /day.(LD50 for levamisole in mice is I50 mg/kg body weight (Bullock, Hand & Waletzky, 1968)). The amount of levamisole consumed by the mice was calculated daily from the amount of medicated water drunk, and adjustments of concentration made from a stock solution (7.5 w/v buffered solution of levamisole hydrochloride provided by Ethnor Pty Ltd). From the first day of infection, different groups of mice were given mebendazole and/or levamisole in their diet. The number of days of anthelmintic treatment was varied. From the day that treatment ceased to the day of autopsy the mice were fed Micon rat and mouse cubes and tapwater. Control mice were fed on cubes and tapwater throughout the experimental period. At the end of the experiments larvae were recovered by peritoneal lavage and trypsin digestion of the liver (Specht & Voge, 1965) and counted, Additional experimental details are given in Tables 14. The results were analysed by the Wilcoxon two sample test for unpaired samples. The effects of the anthelmintics were also tested in vitro. Larvae were isolated aseptically, washed five times in sterile Hank’s solution, and 10 larvae were placed in each of a number of sterile culture bottles containing 8 ml NCTC 135 medium (G&co) with 500
463
464
EVA-MARIA
units penicillin, 500 pg streptomycin mycostatin/ml and 2 ml inactivated
BENNET, CAROLYN
and 100 units of
foetal calf serum (CSL, Melbourne). Where present, mebendazole was added to give a final concentration of 0.04 % using Tween susoending agent. Levamisole was added directly to the medium to gibe a con~nt~tion of 10 mg/l. The
80 as a
oH was adiusted with i-4”/, NaHCO,. changed every 2 days a&the twice daily.
The media were
cultures were examined
BEHM and C. BRYANT
the control. to continue
In one case, the infection
was allowed
for 25 days, with levamisole administration each day. There was still no effect. When mebendazole and levamisole were given
simultaneously, they proved more potent than treatment with mebendazole alone (Table 2). in the absence of treatment there was a 4-fold increase in larval numbers. Four days’ treatment with MB2 reduced the parasite count by about one half of the original inoculation at autopsy (7 days post-infection). Levamisole, with or without prefeeding, had no effect. SimuItaneous administration of meben-
VOL. 8. 1978
TABLE ~-THE EFFECTOF MEBENDAZOLE AND LEVAMISOLE ON THE RECOVERYOF Mesocestoides LARVAEFROMEXPERIMENTALLYINFECTEDMICE No. of days of
drug administration -~-~---_ MBZ 0
Total mg drug consumed/mouse
---. 0 3.5
1 : 4 25 0 10
RESULTS In vitro, control larvae remained normal and active for about seven days, when a small proportion showed breaks in the tegument and became less active. Ten to twenty per cent had died by the 14th day while the remainder appeared healthy for the duration of the experiment (21 days). In mebendazole, larvae were normal and active for five days, but theD started to shrink and display reduced activity. Fifty per cent mortality occurred after 14 days; all were dead after 17 days.The initial reaction to levamisole (10 mg/l) was an incomplete paralysis which passed off after about 14 h. Larvae became less active, compared with controls, and all died within 3 weeks. At higher concentrations of ievamisole (100 mg/l) paralysis persisted for at least 2 weeks, when death ensued; however, within this time the larvae recovered if returned to drug-free medium. When mebendazole and levamisole (10 mg/l) were included in the medium, the mortality curve resembled that due to mebendazole alone, but with the added complication of an initial paralysis followed by deparalysis. Zn viva, however, only mebendazole was effective in removing the infection of M. corti from the mouse (Table 1). A minimum of 5 days’ administration of the drug from the time of infection was necessary to eradicate the larvae, as determined at autopsy on the 10th day of infection. In the absence of any treatment, the number of larvae increased by a factor of 3.5 within 10 days. Exposure to the drug for 1 day effectively inhibited multiplication, but it appeared that 4 days’ exposure was required before individuals of the initial infection disappeared. Levamisole was without effect. Administration of the drug for the full 10 days of infection, and for the 7 days prior to infection, produced larval recoveries which were not significantly different from
UP.
LMS
No. of larvae/ mouse (mean * s.E.) 179 & 25-64 f 13
;:; 14.3 ’ 16 0 I.82
56 + 57 i 16 14 27i 4 0 287 rf 44 311 *71
Groups of 6 mice were infected with 50 larvae/mouse. Mebendazole or Ievamisole was administered in their diet for up to 10 days of infection. On the 10th day, all
mice were killed, and the larvae in livers and peritoneal cavities removed for counting. NB. Adminjstration of LMS for 7 days before infection, so that total drug consumed per mouse exceeded 3-5 mg, had no effect on larval growth arid multiplication. Abbreviations used in Tables: MBZ, mebendazole; LMS, levamisole.
dazole and levamisole. however, produced further significant reduction of larval numbers, to about one tenth of the initial burden. Prefeeding with levamisole did not produce a greater effect. Two experiments were performed to determine the nature of the enhanced effect of mebendazole in the presence of levamisole. Mice were pretreated with heat-killed larvae 7 days before infection and the commencement of anthelmintic administration. TABLE Z-THE EFFECT OF SIMULTANEOUS ADMINISTRATION OF MEBENDAZOLE AND LEVAMISOLE ON THE RECOVERY OF Mesocestoides LARVAE FROM EXPERIMENTALLY INFECTED MICE
Total mg drug consumed/mouse MBZ LMS
No, of larvae/ mouse (mean i SE.‘; I. none 202 * 89 10 2. MBZ 9.4 28&18 9 I& 192+30 5 3. LMS LMS *1..5 + 0.8 232 + 30 5 4. 8.0 6 *- 7t 4 5. MBZ + LMS 0.35 MBZ + LMS IO.4 *I‘S +- 0.5 6. 8+5$ __-__ -_5 Groups of mice were infected with 50 larvae/mouse. Mebendazole or levamisole, or both drugs, were administered in their diet for 4 days. On the 7th day of Drug
infection all mice were killed, and treated as before. *Amount of LMS consumed in 7 days preceding infection. ~Signi~cantly different from treatment with MBZ alone
P = 0.01.
ISignificantly
different
from
treatment
with
MBZ
alone P < 0.05.
Table 3 shows once again that, in the controls, combined
administration
of mebendazole
and leva-
I.J.P. VOL.
Anthelmintics
8. 1978
and Mesocestoides
465
corti in mice
TABLE ~--THE EFFECT 0~ PREVIOUS ADMINISTRATION OF 100 mg HEATINACTlVATEDLARVAEONTHERESPONSETOMEBENDAZOLEORLEVAMISOLE OF Mesocestoides LARVAL GROWTH IN EXPERIMENTALLY INFECTED MICE
Drug
Controls
Treated with inactivated larvae
Treated with inactivated larvae; prefed LMS for 7 days (total dose 1.1 mg/mouse) 11* 3* (6.2) 187 & 14* (0.5) 5zt 1*t (5.3; 0.3)
None 323 i 23 157 i 11* MBZ 64i 3 II& 3* (dose/mouse in mg) (5.8) (4.6) 285 i_ I8 I75 f 15* LMS (dose/mouse in mg) (0.5) (0.5) MBZ + LMS 34 f 6P 13& 6* (4.9; 0.3) (dose/mouse in mg) (5.5; 0.3) Groups of IO mice received 100 mg heat inactivated larvae in 0.85% NaCl by intraperitoneal injection. Control groups received only NaCI. All groups were subsequently infected with 50 larvae/ mouse and then were treated for 3 days as shown in the table. On the 7th day of infection all mice were killed and larval counts made as before. *Significantly different from the appropriate control at the level P < 0.005. tsignificantly different from the group treated with inactivated larvae P < 0.005; P significantly different from control group treated with MBZ, alone, P < 0.005.
misole was more effective than mebendazole alone. Treating with dead larvae was also effective in reducing worm count; only about half the number of larvae were recovered compared with the control. When mebendazole was given to this group, worm recovery was depressed well below that of the corresponding control. Levamisole alone had no effect, neither was there an additional effect due to the drugs in combination, except possibly when the mice had received levamisole for 7 days prior to infection. In the second experiment, mice were irradiated 3 days before commencing the anthelmintic regime (Table 4). Unirradiated controls behaved as described earlier. In irradiated mice, mebendazole was less effective than in the control in reducing worm burden possibly because of reduced consumption of feed; and simultaneous administration of levamisole had no effect.
DISCUSSION
Oral administration of mebendazole to mice infected with M. corti tetrathyridia is highly effective in removing the infection. Levamisole, administered orally, is ineffective. A combination of these two anthelmintics produces a synergistic effect; that is, together the drugs are more effective than when either is administered alone. Dead larvae, if injected into mice prior to infection, confer partial immunity on the host (Kazacos, 1976), and additional mebendazole treatment results in early cure. However, the synergistic effect of leva-
TABLE ~-THE EFFECT OF IRRADIATION ON THE RESPONSE TOMEBENDAZOLEORLEVAM~~~LEOF Mesocestoides LARVAL GROWTH IN EXPERIMENTALLYINFECTED MICE
Drug
Larval Recovery/Mouse Control Irradiated
None MBZ (dose/mouse in mg) LMS (dose/mouse in mg) MBZ + LMS (dose/mouse in mg)
213 f 21 23i 5 (7.2) 224 + 13 (0.5) 9+ 3’ (5.1; 0.5)
170 i 10 68 * 7t (4.6) 197 -I 30 (0.3) 74 * 15P (3.7; 0.3)
Mice were given a dose of 750 rads from a Wo source, and left to recover for 3 days. Groups of 10 were then infected with 50 larvae/mouse and treated for 3 days as shown in the Table. On the 7th day all mice were killed and larval counts made as before. Control groups were also treated. *Significantly different from the control receiving only MBZ, P < 0.01. tsignificantly different from the appropriate control P < 0.005. misole is lost. Similarly, in mice whose immune system has been destroyed by irradiation, the drugs do not interact synergistically and mebendazole alone is less effective. These experiments suggest that mebendazole acts in the following way. At low doses tetrathyridial multiplication is suppressed, but the injected larvae which comprise the infection are not removed. Higher doses damage or kill larvae; parasite material then activates the immune system which, in conjunc-
466
EVA-MARIA BENNET,CAROLYNBFHM and C. BRYANT
tion with the drug, destroys the remaining larvae. The synergistic effect of levamisole may then be ascribed to a non-specific adjuvant effect on the immune system. Destruction of this system or its preliminary specific stimulation with dead worm material thus abolishes the effect of levamisole. This conclusion is supported by the observation that, in vitro, the combined effects of mebendazole and levamisole are not materially different from those due to mebendazole alone. Theseexperiments should, however, be interpreted with caution as it is clear that, in vitro levamisole by itself eventually brings about the deaths of the larvae, probably in a similar manner to that described for nematodes by Coles, East & Jenkins (1974). The fact that levamisole is ineffective against the larvae in vivo could be explained by the position of the worms in the host. Located in the liver and peritoneal cavity, the larvae will remain there even when exposed to and paralysed by levamisole. As levamisole is cleared from the blood, the larvae recover, as larvae maintained in vitro recovered when removed to fresh medium. In contrast, gastrointestinal and pulmonary nematodes exposed to and paralysed by levamisole in vivo will be expelled from the intestine and lungs of the host. The fact that levamisole enhances the activity of mebendazole in vivo may be of commercial importance if the effect extends also to domestic animals, as its inclusion in formulations of the rather expensive benzimidazoles may permit a lower dose-rate and cheaper drench. Acknowledgement--The
authors thank Mr R. A. F. Chevis of Ethnor Pty Ltd, Australia for much helpful advice on the administration of drugs and for the supply of mebendazole feed and levamisole.
I.J.P. VOL.8. 1978
REFERENCES BULLOCK M. W., HAND J. J. & WALE~ZKY E. 1968. Resolution and racemization of dl-tetramisole, dl-6phenyl-2,3,5,6 tetrahydroimidazo-(2,1-b) thiazole. Journal of Medical Chemistry 11: 169-l 7 1. COLES G. C. 1974. The reversible in vitro paralysis of nematodes by levamisole. Parasitology 69: (2) xxii. COLES G. C., EASTJ. M. &JENKINS S. M. 1974. Mode of action of four anthelmintics. Experientia 30: 12651266.
HEATH D. D., CHRISTIEM. J. & CHEVISR. A. F. 1975. The lethal effects of mebendazole on secondary Echinococcus granufosus, cysticerci of Taenia pisiformis and tetrathyridia of Mesocestoides corti. Parasitology 70: 273-285.
HOEBKEJ. & FRANCHIG. 1973. Influence of tetramisole and its optical isomers on the mononuclear phagocytic system. Journal of the Reticuloendothelial Society 14: 317-377.
KAZACOS K. R. 1976. Immunization of mice against Mesocestoides corti by subcutaneous inoculation of living tetrathyridia. Journal of Parasitology 62: 161163.
MARSBOOMR. 1973. Toxicity studies on mebendazole. Toxicology and Applied Pharmacology
24: 371-377.
RENOUXG. & RENOUXM. 1974. Modulation of immune reactivity by phenylimidothiazole salts (tetramisole and levamisole) in mice immunized by sheep red blood cells. Journal of Immunology 113: 779-790. SPECHTD. & VOGE M. 1965. Asexual multiplication of Mesocestoides tetrathyridia in laboratory animals. Journal of ParasitoloFv 51: 268-272.
SPECHT D..& WIDMER% A. 1972. Response of mouse liver to infection with tetrathyridia of Mesocestoides (Cestoda). Journal of Parasitology 58: 431-437. VAN DEN BOSSCHEH. & JANSSENP. A. J. 1969. The biochemical mechanism of action of the drug tetramisole. Biochemical Pharmbcology 18, 35-42.
of Zoology, Australian National University, Canberra, Australia (Received 20 December
1977)
Abstract-BENNET E.-M., BEHM C. A. and BRYANTC. 1978. Effects of mebendazole and levamisole on tetrathyridia of Mesocestoides corti in the mouse. International Journal for Parasitology 8: 463-466.
Mebendazole, but not levamisole, administered to mice carrying artificial infections of 50 tetrathyridia of Mesocestoides corti, was effective in killing the parasites. However, simultaneous administration of mebendazole and levamisole was still more effective. Treatment with levamisole before infection had no additional effect. Injection of mice with dead larvae offered some protection against a subsequent challenge with 50 live larvae; however, levamisole did not then improve the anthelmintic efficacy of mebendazole. In mice rendered immunoincompetent by radiation mebendazole was less effective than in nonirradiated controls and levamisole again did not enhance the effect of mebendazole. It is concluded that anthelmintic efficacy of mebendazole depends on its anthelmintic activity supplemented by the host’s immune response; and that levamisole stimulates the latter. 1NDEX KEY WORDS: anthelmintics;
mebendazole;
INTRODUCTION
SPECHT & VOGE (1965) found that tetrathyridia of Mesocestoides corti (Cestoda: Cyclophyllidea) multiply asexually in the liver and body cavity of mice
and can be serially transferred by intraperitoneal inoculation. Destruction of the liver by the larvae initially causeq a non-specific inflammatory response. Later, specific resistance to the parasite develops, which limits parasitic multiplication but does not prevent the eventual death of the mouse (Specht & Widmer, 1972). Immunity to tetrathyridia is both cellular and humoral (Pollacco, Nicholas, Mitchell and Stewart, 1978, submitted for publication). Mebendazole is a broad-spectrum anthelmintic, effective against adult cestodes and also capable of killing larva1 cestodes, including M. corti (Heath, Christie & Chevis, 1975). Levamisole is effective against nematodes at low concentrations (Van den Bossche & Janssen, 1969) but appears to be without action against cestodes. Its primary effect seems to be exerted on the nematode nervous system (Coles, 1974). Levamisole has also attracted attention because it stimulates the immune response (Renoux & Renoux, 1974). How it acts as an immunoadjuvant is unknown. In mice it is likely that the macrophage system is involved (Hoebke & Franchi, 1973). This paper describes the effect of levamisole in enhancing the anthelmintic activity of mebendazole against tetrathyridia of M. corti in mice. MATERIALS
AND
METHODS
A strain of M. corti, originally isolated by Specht &
levamisole; Mesocestoides corti.
Voge (I 965), was maintained by serial passage in a closed colony of outbred mice (Piebald strain). Only female mice were used in experiments: their ages ranged from 5 to 8 weeks at infection. Mebendazole was administered as a 1 g/kg constituent of a peanut meal formulated by Ethnor Pty Ltd, which formed the sole diet of the mice for the duration of the experiment. Such administration approximated a dose of 90-250 mg/kg body weight/day and falls well below the LDSOfor mice, which exceeds I g/kg body weight (Marsboom, 1973). The amount of mebendazole consumed by the mice was determined daily by weighing the medicated feed. Levamisole was administered in drinking water at a level which approximated a dose of IO mg/kg body weight /day.(LD50 for levamisole in mice is I50 mg/kg body weight (Bullock, Hand & Waletzky, 1968)). The amount of levamisole consumed by the mice was calculated daily from the amount of medicated water drunk, and adjustments of concentration made from a stock solution (7.5 w/v buffered solution of levamisole hydrochloride provided by Ethnor Pty Ltd). From the first day of infection, different groups of mice were given mebendazole and/or levamisole in their diet. The number of days of anthelmintic treatment was varied. From the day that treatment ceased to the day of autopsy the mice were fed Micon rat and mouse cubes and tapwater. Control mice were fed on cubes and tapwater throughout the experimental period. At the end of the experiments larvae were recovered by peritoneal lavage and trypsin digestion of the liver (Specht & Voge, 1965) and counted, Additional experimental details are given in Tables 14. The results were analysed by the Wilcoxon two sample test for unpaired samples. The effects of the anthelmintics were also tested in vitro. Larvae were isolated aseptically, washed five times in sterile Hank’s solution, and 10 larvae were placed in each of a number of sterile culture bottles containing 8 ml NCTC 135 medium (G&co) with 500
463
464
EVA-MARIA
units penicillin, 500 pg streptomycin mycostatin/ml and 2 ml inactivated
BENNET, CAROLYN
and 100 units of
foetal calf serum (CSL, Melbourne). Where present, mebendazole was added to give a final concentration of 0.04 % using Tween susoending agent. Levamisole was added directly to the medium to gibe a con~nt~tion of 10 mg/l. The
80 as a
oH was adiusted with i-4”/, NaHCO,. changed every 2 days a&the twice daily.
The media were
cultures were examined
BEHM and C. BRYANT
the control. to continue
In one case, the infection
was allowed
for 25 days, with levamisole administration each day. There was still no effect. When mebendazole and levamisole were given
simultaneously, they proved more potent than treatment with mebendazole alone (Table 2). in the absence of treatment there was a 4-fold increase in larval numbers. Four days’ treatment with MB2 reduced the parasite count by about one half of the original inoculation at autopsy (7 days post-infection). Levamisole, with or without prefeeding, had no effect. SimuItaneous administration of meben-
VOL. 8. 1978
TABLE ~-THE EFFECTOF MEBENDAZOLE AND LEVAMISOLE ON THE RECOVERYOF Mesocestoides LARVAEFROMEXPERIMENTALLYINFECTEDMICE No. of days of
drug administration -~-~---_ MBZ 0
Total mg drug consumed/mouse
---. 0 3.5
1 : 4 25 0 10
RESULTS In vitro, control larvae remained normal and active for about seven days, when a small proportion showed breaks in the tegument and became less active. Ten to twenty per cent had died by the 14th day while the remainder appeared healthy for the duration of the experiment (21 days). In mebendazole, larvae were normal and active for five days, but theD started to shrink and display reduced activity. Fifty per cent mortality occurred after 14 days; all were dead after 17 days.The initial reaction to levamisole (10 mg/l) was an incomplete paralysis which passed off after about 14 h. Larvae became less active, compared with controls, and all died within 3 weeks. At higher concentrations of ievamisole (100 mg/l) paralysis persisted for at least 2 weeks, when death ensued; however, within this time the larvae recovered if returned to drug-free medium. When mebendazole and levamisole (10 mg/l) were included in the medium, the mortality curve resembled that due to mebendazole alone, but with the added complication of an initial paralysis followed by deparalysis. Zn viva, however, only mebendazole was effective in removing the infection of M. corti from the mouse (Table 1). A minimum of 5 days’ administration of the drug from the time of infection was necessary to eradicate the larvae, as determined at autopsy on the 10th day of infection. In the absence of any treatment, the number of larvae increased by a factor of 3.5 within 10 days. Exposure to the drug for 1 day effectively inhibited multiplication, but it appeared that 4 days’ exposure was required before individuals of the initial infection disappeared. Levamisole was without effect. Administration of the drug for the full 10 days of infection, and for the 7 days prior to infection, produced larval recoveries which were not significantly different from
UP.
LMS
No. of larvae/ mouse (mean * s.E.) 179 & 25-64 f 13
;:; 14.3 ’ 16 0 I.82
56 + 57 i 16 14 27i 4 0 287 rf 44 311 *71
Groups of 6 mice were infected with 50 larvae/mouse. Mebendazole or Ievamisole was administered in their diet for up to 10 days of infection. On the 10th day, all
mice were killed, and the larvae in livers and peritoneal cavities removed for counting. NB. Adminjstration of LMS for 7 days before infection, so that total drug consumed per mouse exceeded 3-5 mg, had no effect on larval growth arid multiplication. Abbreviations used in Tables: MBZ, mebendazole; LMS, levamisole.
dazole and levamisole. however, produced further significant reduction of larval numbers, to about one tenth of the initial burden. Prefeeding with levamisole did not produce a greater effect. Two experiments were performed to determine the nature of the enhanced effect of mebendazole in the presence of levamisole. Mice were pretreated with heat-killed larvae 7 days before infection and the commencement of anthelmintic administration. TABLE Z-THE EFFECT OF SIMULTANEOUS ADMINISTRATION OF MEBENDAZOLE AND LEVAMISOLE ON THE RECOVERY OF Mesocestoides LARVAE FROM EXPERIMENTALLY INFECTED MICE
Total mg drug consumed/mouse MBZ LMS
No, of larvae/ mouse (mean i SE.‘; I. none 202 * 89 10 2. MBZ 9.4 28&18 9 I& 192+30 5 3. LMS LMS *1..5 + 0.8 232 + 30 5 4. 8.0 6 *- 7t 4 5. MBZ + LMS 0.35 MBZ + LMS IO.4 *I‘S +- 0.5 6. 8+5$ __-__ -_5 Groups of mice were infected with 50 larvae/mouse. Mebendazole or levamisole, or both drugs, were administered in their diet for 4 days. On the 7th day of Drug
infection all mice were killed, and treated as before. *Amount of LMS consumed in 7 days preceding infection. ~Signi~cantly different from treatment with MBZ alone
P = 0.01.
ISignificantly
different
from
treatment
with
MBZ
alone P < 0.05.
Table 3 shows once again that, in the controls, combined
administration
of mebendazole
and leva-
I.J.P. VOL.
Anthelmintics
8. 1978
and Mesocestoides
465
corti in mice
TABLE ~--THE EFFECT 0~ PREVIOUS ADMINISTRATION OF 100 mg HEATINACTlVATEDLARVAEONTHERESPONSETOMEBENDAZOLEORLEVAMISOLE OF Mesocestoides LARVAL GROWTH IN EXPERIMENTALLY INFECTED MICE
Drug
Controls
Treated with inactivated larvae
Treated with inactivated larvae; prefed LMS for 7 days (total dose 1.1 mg/mouse) 11* 3* (6.2) 187 & 14* (0.5) 5zt 1*t (5.3; 0.3)
None 323 i 23 157 i 11* MBZ 64i 3 II& 3* (dose/mouse in mg) (5.8) (4.6) 285 i_ I8 I75 f 15* LMS (dose/mouse in mg) (0.5) (0.5) MBZ + LMS 34 f 6P 13& 6* (4.9; 0.3) (dose/mouse in mg) (5.5; 0.3) Groups of IO mice received 100 mg heat inactivated larvae in 0.85% NaCl by intraperitoneal injection. Control groups received only NaCI. All groups were subsequently infected with 50 larvae/ mouse and then were treated for 3 days as shown in the table. On the 7th day of infection all mice were killed and larval counts made as before. *Significantly different from the appropriate control at the level P < 0.005. tsignificantly different from the group treated with inactivated larvae P < 0.005; P significantly different from control group treated with MBZ, alone, P < 0.005.
misole was more effective than mebendazole alone. Treating with dead larvae was also effective in reducing worm count; only about half the number of larvae were recovered compared with the control. When mebendazole was given to this group, worm recovery was depressed well below that of the corresponding control. Levamisole alone had no effect, neither was there an additional effect due to the drugs in combination, except possibly when the mice had received levamisole for 7 days prior to infection. In the second experiment, mice were irradiated 3 days before commencing the anthelmintic regime (Table 4). Unirradiated controls behaved as described earlier. In irradiated mice, mebendazole was less effective than in the control in reducing worm burden possibly because of reduced consumption of feed; and simultaneous administration of levamisole had no effect.
DISCUSSION
Oral administration of mebendazole to mice infected with M. corti tetrathyridia is highly effective in removing the infection. Levamisole, administered orally, is ineffective. A combination of these two anthelmintics produces a synergistic effect; that is, together the drugs are more effective than when either is administered alone. Dead larvae, if injected into mice prior to infection, confer partial immunity on the host (Kazacos, 1976), and additional mebendazole treatment results in early cure. However, the synergistic effect of leva-
TABLE ~-THE EFFECT OF IRRADIATION ON THE RESPONSE TOMEBENDAZOLEORLEVAM~~~LEOF Mesocestoides LARVAL GROWTH IN EXPERIMENTALLYINFECTED MICE
Drug
Larval Recovery/Mouse Control Irradiated
None MBZ (dose/mouse in mg) LMS (dose/mouse in mg) MBZ + LMS (dose/mouse in mg)
213 f 21 23i 5 (7.2) 224 + 13 (0.5) 9+ 3’ (5.1; 0.5)
170 i 10 68 * 7t (4.6) 197 -I 30 (0.3) 74 * 15P (3.7; 0.3)
Mice were given a dose of 750 rads from a Wo source, and left to recover for 3 days. Groups of 10 were then infected with 50 larvae/mouse and treated for 3 days as shown in the Table. On the 7th day all mice were killed and larval counts made as before. Control groups were also treated. *Significantly different from the control receiving only MBZ, P < 0.01. tsignificantly different from the appropriate control P < 0.005. misole is lost. Similarly, in mice whose immune system has been destroyed by irradiation, the drugs do not interact synergistically and mebendazole alone is less effective. These experiments suggest that mebendazole acts in the following way. At low doses tetrathyridial multiplication is suppressed, but the injected larvae which comprise the infection are not removed. Higher doses damage or kill larvae; parasite material then activates the immune system which, in conjunc-
466
EVA-MARIA BENNET,CAROLYNBFHM and C. BRYANT
tion with the drug, destroys the remaining larvae. The synergistic effect of levamisole may then be ascribed to a non-specific adjuvant effect on the immune system. Destruction of this system or its preliminary specific stimulation with dead worm material thus abolishes the effect of levamisole. This conclusion is supported by the observation that, in vitro, the combined effects of mebendazole and levamisole are not materially different from those due to mebendazole alone. Theseexperiments should, however, be interpreted with caution as it is clear that, in vitro levamisole by itself eventually brings about the deaths of the larvae, probably in a similar manner to that described for nematodes by Coles, East & Jenkins (1974). The fact that levamisole is ineffective against the larvae in vivo could be explained by the position of the worms in the host. Located in the liver and peritoneal cavity, the larvae will remain there even when exposed to and paralysed by levamisole. As levamisole is cleared from the blood, the larvae recover, as larvae maintained in vitro recovered when removed to fresh medium. In contrast, gastrointestinal and pulmonary nematodes exposed to and paralysed by levamisole in vivo will be expelled from the intestine and lungs of the host. The fact that levamisole enhances the activity of mebendazole in vivo may be of commercial importance if the effect extends also to domestic animals, as its inclusion in formulations of the rather expensive benzimidazoles may permit a lower dose-rate and cheaper drench. Acknowledgement--The
authors thank Mr R. A. F. Chevis of Ethnor Pty Ltd, Australia for much helpful advice on the administration of drugs and for the supply of mebendazole feed and levamisole.
I.J.P. VOL.8. 1978
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HEATH D. D., CHRISTIEM. J. & CHEVISR. A. F. 1975. The lethal effects of mebendazole on secondary Echinococcus granufosus, cysticerci of Taenia pisiformis and tetrathyridia of Mesocestoides corti. Parasitology 70: 273-285.
HOEBKEJ. & FRANCHIG. 1973. Influence of tetramisole and its optical isomers on the mononuclear phagocytic system. Journal of the Reticuloendothelial Society 14: 317-377.
KAZACOS K. R. 1976. Immunization of mice against Mesocestoides corti by subcutaneous inoculation of living tetrathyridia. Journal of Parasitology 62: 161163.
MARSBOOMR. 1973. Toxicity studies on mebendazole. Toxicology and Applied Pharmacology
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RENOUXG. & RENOUXM. 1974. Modulation of immune reactivity by phenylimidothiazole salts (tetramisole and levamisole) in mice immunized by sheep red blood cells. Journal of Immunology 113: 779-790. SPECHTD. & VOGE M. 1965. Asexual multiplication of Mesocestoides tetrathyridia in laboratory animals. Journal of ParasitoloFv 51: 268-272.
SPECHT D..& WIDMER% A. 1972. Response of mouse liver to infection with tetrathyridia of Mesocestoides (Cestoda). Journal of Parasitology 58: 431-437. VAN DEN BOSSCHEH. & JANSSENP. A. J. 1969. The biochemical mechanism of action of the drug tetramisole. Biochemical Pharmbcology 18, 35-42.
