VETERINARSKI ARHIV 69 (1),
29-37, 1999

ISSN 1331-8055 Online
ISSN
0372-5480 Printed in Croatia

Reduction of dichlorvos-induced
toxicosis in
rabbits by levamisole

Imad Ibrahim Aldabagh and Fouad
Kasim Mohammad*

Department of Physiology,
Biochemistry and Pharmacology,
College of Veterinary Medicine, University of
Mosul, Iraq

* Contact address:
Prof. Dr. Fouad
Kasim Mohammad,
Department of Physiology, Biochemistry and Pharmacology,
College of Veterinary Medicine, University of Mosul, P.O. Box 11136, Mosul, Iraq

ALDABAGH, I. I., F. K. MOHAMMAD:
Reduction of dichlorvos-induced toxicosis in rabbits by levamisole. Vet. arhiv
69, 29-37, 1999.

ABSTRACT

The toxicity and interaction of
levamisole with dichlorvos were examined in male rabbits. Signs of levamisole
(25, 50 and 75 mg/kg, subcutaneously) poisoning in rabbits were dose-dependent,
and the highest dose produced excessive salivation, lacrimation, ataxia and
tremors in all the rabbits. This dose induced convulsion and death in 50 and 75%
of the rabbits, respectively. Levamisole at 25 and 75% did not significantly
affect erythrocyte acetylcholinesterase (EChE) activity at 2 and 24 h after
treatment. Dichlorvos dosing at 10 and 20 mg/kg, orally dose-dependently induced
signs of cholinergic toxicity. The 20 mg/kg dose of dichlorvos caused death in
75% of the rabbits. Levamisole pretreatment at 50 mg/kg protected the rabbits
against dichlorvos (20 mg/kg, orally) induced toxicosis, prevented the
occurrence of convulsion, gasping and death, and significantly increased the
latency to onset of tremor in comparison with the dichlorvos-treated control
group. Combined levamisole (50 mg/kg) and dichlorvos (10 mg/kg) treatments
caused a significantly higher degree of EChE inhibition at 2 and 24 h
post-treatment in comparison with the dichlorvos-treated group. The data suggest
that levamisole protects rabbits intoxicated with dichlorvos, irrespective of
the extent of EChE inhibition.

Key words: levamisole,
dichlorvos, rabbit, erythrocyte cholinesterase symptoms, organophosphate

Introduction

Levamisole, the 1-isomer of
tetramisole, is an anthelmintic used in veterinary practice (MARRINER and
ARMOUR, 1986; DALVI, 1990). Levamisole poisoning in animals is characterised by
salivation, lacrimation, miosis, hyperventilation or dyspnea, frequent urination
and defecation, ataxia, tremors and convulsion before death (HSU, 1980, 1981a,
1981b; DALVI, 1990). These signs of levamisole poisoning resemble those of
organophosphorus (OP) insecticides which cause muscarinic, nicotinic and central
nervous system effects (ANON., 1986; FIKES, 1990). Thus, it appears that there
is the possibility of potentiated toxic effects when levamisol and OP
insecticides are concurrently used in animals.

Treatment of mice (HSU, 1980) and pig
(HSU, 1981a) with the PO insecticide dichlorvos did not increase the toxicity of
levamisole. Similarly, the PO insecticide diazinon did not increase the toxicity
of tetramisole in sheep (FORSYTH, 1966). However, in these studies the
insecticides were given prior to levamisole (HSU, 1980, 1981a) or tetramisole
(FORSYTH, 1966). The present study was undertaken in rabbits in order to further
examine levamisole-induced toxicosis and its interaction with dichlorvos, taking
account of the administration of levamisole before dichlorvos dosing. This
approach provided some new information about the ability of levamisole in
reducing the toxic effects of dichlorvos.

Materials and methods

Male cross-bred rabbits with masses of
1.2-1.9 kg were used. They were housed at a temperature of 22±2 °C with 10/14 h
light/dark cycle. The animals had free access to food and water.

Experiment I, toxic effects of
levamisole:

Rabbits (n=4/group) were treated
subcutaneously (s.c.) with levamisole hydrochloride (7.5%, Phenex, Belgium) at
dose rates of 25, 50 and 75 mg/kg body mass. The control group received
physiological saline solution at 1 ml/kg, s.c. Levamisole doses were
determined in a preliminary experiment in rabbits. After levamisole injection,
each rabbit was observed until death, or for 2 h, for the appearance of signs of
poisoning which included salivation, lacrimation, frequent urination and
defecation, ataxia, tremor and convulsion.

Two and 24 h after levamisole
administration (25 and 50 mg/kg, s.c.) blood samples were obtained from the ear
vein by a heparinised syringe. Erythrocytes were separated by centrifugation of
the blood samples at 3000 rpm for 15 minutes. An aliquot of 0.1 ml of
erythrocytes was used for the determination of erythrocyte acetylcholinesterase
(EChE) activity by the modified electrometric method described previously
(MOHAMMAD and OMER, 1982).

Experiment II, effects of levamisole
pre-treatment on dichlorvos-induced toxicosis:

In this experiment, 4 rabbits/group
were used. Levamisole was administered at dose rates of 0 (saline-control), 25
and 50 mg/kg, s.c. 30 minutes before dosing of the rabbits with dichlorvos
(Aforfos 50% EC, K.N. Efthymiadis, Greece) at 10 mg/kg, orally. Also, rabbits
were treated with levamisole at 0 (saline-control) and 50 mg/kg, s.c. 30 minutes
before dosing with a higher dose of dichlorvos (20 mg/kg, orally). The doses of
dichlorvos were predetermined to produce cholinergic toxicity in rabbits.
Dichlorvos was diluted with distilled water and administered by a gavage needle
to a volume of 0.5 ml/kg body mass. Following dichlorvos dosing, each rabbit was
observed until death, or for 2 h, for the occurrence of signs of toxicosis
characteristic of cholinergic hyperstimulation (ANON., 1986; FIKES, 1990; FARIS
and MOHHAMAD, 1997). The severity score of toxicity in rabbits doses with
dichlorvos was determined by adding the assigned grades to the signs of
toxicosis: 0=normal, 1=hyperactivity and/or agitation, 2=salivation, 3=tremor,
4=muscle fasciculation, 5=flat body appearance, 6=convulsion, 7=death.

The latencies to onset of tremor and
death within 2 h were recorded. The activity of EChE was determined as mentioned
in experiment I prior to drug administration (time 0, baseline) and thereafter 2
and 24 h after dichlorvos dosing at 10 mg/kg. The percent of EChE inhibition was
determined as follows:

% inhibition= SEChE activity (baseline)-EChE activity at 2 or 24
h/EChE activity (baseline)C × 100

In both experiments, the rabbits were
retained for 3 days for any delayed death.

Statistics: Frequency data were
subjected to Fisher's exact probability test (RUNYON, 1977). The severity score
to toxicity was analysed by Mann-Whitney-U-test (RUNYON, 1977). Tremor times
were compared by Unpaired Student's-t-test (BRUNING and KINTZ, 1977). The level
of significance was at P<0.05.

Results

Subcutaneous injection of levamisole in
rabbits induced signs of poisoning that were dose dependent (Table 1). At the
highest dose (75 mg/kg) tested, levamisole significantly increased the
occurrence of excessive salivation, lacrimation, ataxia and tremor by 100%, and
caused convulsion and death in 50 and 75% of the rabbits, respectively (Table
1). The mean±SE latency to onset of death was 55±20 minutes. The 50 mg/kg dose
of levamisole was well tolerated by the rabbits which showed salivation (75%),
lacrimation (50%) as well as ataxia and tremor (25%) in comparison with the
control group (Table 1). Levamisole at 25 and 50 mg/kg did not significantly
affect EChE activity at 2 and 24 h after treatment (Table 2). However, in the
levamisole-treated rabbits EChE activity was decreased by 16-18%, at 2 h
post-treatment, in comparison with the control value (Table 2).

Table 1. Levamisole-induced toxicosis
in rabbits

Variable
(% occurrence)

Control

Levamisole (mg/kg, s.c.)

25

50

75

Salivation

0

25

75

100*

Lacrimation

0

0

50

100*

Urination

25

25

0

0

Defecation

25

75

25

100

Ataxia

0

0

25

100*

Tremor

0

0

25

100*

Convulsion

0

0

0

50

Death

0

0

0

75

N=4 rabbits/dose group. Each rabbit was
observed until death or for 2 h after levamisole treatment.
*Significantly
different from the corresponding control value, P<0.05.

Table 2. Erythrocyte
acetylcholinesterase activity in rabbits treated with levamisole

Levamisole
(mg/kg, s.c.)

Difference pH / 30 minutes

2 h

24 h

0 (saline)

0.45±0.02

0.39±0.04

25

0.37±0.03

0.39±0.06

50

0.38±0.05

0.37±0.06

Dichlorvos dosing at 10 or 20 mg/kg in
rabbits dose-dependently induced signs of toxicosis characteristics of
cholinesterase inhibition and subsequent cholinergic overstimulation (Table 3).
These signs were salivation, lacrimation, frequent urination and defecation,
tremor, ataxia, muscle fasciculation, flat body appearance, gasping and
convulsion (Table 3). The 20 mg/kg dose of dichlorvos caused death in 3 out of 4
rabbits. The latency to onset of death was 25±7 minutes.

Table 3. Effect of levamisole on
dichlorvos-induced toxicosis in rabbits

Variable
(% occurrence)

Dichlorvos (mg/kg, orally)

10

20

Levamisole (mg/kg, s.c.)

0

25

50

0

50

Salivation

100

100

100

100

100

Lacrimation

100

100

100

100

100

Urination

75

75

25

100

25

Defecation

75

100

50

50

50

Ataxia

100

100

100

100

100

Tremor

100

100

100

100

100

Muscle fasciculation

100

25

25

100

50

Flat body appearance

100

75

25

100

50

Convulsion

0

0

0

75

0

Gasping

0

0

0

75

0

Death

0

0

0

75

0

Median severity score*

15

11*

8*

28

10.5*

Rabbits (4/group) were pretreated with
levamisole 30 minutes before dichlorvos dosing. Each rabbit was observed until
death or for 2 h after dichlorvos dosing. *The severity score of toxicity for
each rabbit was the summation of grades of sign of toxicosis from 0 to 7:
0=normal; 1=hyperactivity and/or agitation; 2=salivation; 3=tremor; 4=muscle
fasciculation; 5=flat body appearance; 6=convulsion; 7=death. For reference, the
highest severity score when all the signs of toxicosis mentioned above are
observed in an individual rabbit would be 28. *Significantly different from the
respective control (0 dose) value, P<0.05.

Levamisole pre-treatment protected the
rabbits against dichlorvos (10 or 20 mg/kg) induced toxicosis, and the median
score of the severity of toxicosis significantly decreased in comparison with
the respective control non-levamisole-treated groups (Table 3). The protective
effect of levamisole was best seen at the dose rate of 50 mg/kg when given
before the high dose (20 mg/kg) of dichlorvos. At these doses of both drugs,
levamisole decreased the occurrence of frequent urination, muscle fasciculation
and flat body appearance, but most importantly, levamisole blocked the
occurrence of convulsion, gasping and death induced by dichlorvos (Table 3).
Levamisole at 50 mg/kg significantly prolonged the latency to onset of tremor in
the dichlorvos-treated group when compared with saline-dichlorvos (10 or 20
mg/kg) treated groups (Table 4).

Table 4. Effect of levamisole on the
latency to onset of tremors in rabbits dosed with dichlorvos

Treatment (mg/kg)

Latency to onset of tremor
(min)

Saline + Dichlorvos (10)
(control)

8.0 ± 1.2

Levamisole (25) + Dichlorvos (10)

9.0 ± 0.7

Levamisole (50) + Dichlorvos (10)

11.8 ± 1.3*

Saline + Dichlorvos (20)
(control)

3.8 ± 0.5

Levamisole (50) + Dichlorvos (20)

8.8 ± 1.0*

The values are mean±SE of 4
rabbits/group. Levamisole was injected subcutaneously 30 minutes before oral
dosing of dichlorvos. *Significantly different from the respective control
value, P<0.05.

Fig. 1. Percentage of erythrocyte
acetylcholinesterase (EChE) inhibition in rabbits dosed orally with dichlorvos
(10 mg/kg) after pre-treatment with saline (control) and levamisole at 25 and 50
mg/kg, s.c.. Pre-treatment (baseline) values (mean±SE) of EChE activity of the
three groups were 0.44±0.02, 0.38±0.03 and 0.41±0.02, difference pH/30 minutes,
respectively. %inhibition=SEChE activity
(baseline)-EChE activity at 2 or 24 h / EChE activity (baseline)C × 100. Levamisole was injected 30 minutes before
dichlorvos dosing. *Significantly different from the control (dichlorvos-saline)
group, P<0.05.

Combined levamisole (50 mg/kg) and
dichlorvos (10 mg/kg) treatments caused a significantly higher degree of EChE
inhibition at 2 and 24 h after dichlorvos dosing in comparison with the
dichlorvos-treated group (Figure 1).

Discussion

Levamisole produced signs of toxicosis
in rabbits characteristic of cholinergic overstimulation. These effects were in
agreement with those reported in sheep treated with tetramisole (FORSYTH, 1966;
PHILIP and STONE, 1967) or pigs and mice treated with levamisole (HSU, 1980;
1981a). The findings of the present study do not support the hypothesis that
levamisole, owing to its cholinomimetic effects, could potentiate the toxicity
of organophosphates. As found in Table 3, levamisole prevented the occurrence of
convulsion, gasping and death in rabbits dosed with dichlorvos at 20 mg/kg. The
results further support the findings of others, who found that there was no
adverse interaction between dichlorvos and levamisole (HSU, 1980; 1981a).
However, our results extend these findings and further suggest that levamisole
possesses a protective effect against dichlorvos-induced toxicosis and lethality
in rabbits. The clinical implication of this study is that levamisole could be
safely used with OP insecticides in animals that need combined anthelmintic and
insecticidal therapy. Further field studies, however, are needed in target
animal species, such as ruminants, to verify and support this point.

The mechanism of the protective effect
of levamisole against dichlorvos-induced toxicosis is not clear at present.
Tetramisole (EYRE, 1970) and levamisole (ALDABAGH and MOHAMMAD, 1998) were found
to inhibit acetylcholinesterase activity in vitro. Considering the fact that
levamisole at 25 and 50 mg/kg decreased (though non-significantly) EChE in the
rabbits by 18 and 16%, respectively (Table 2), it is therefore possible that
levamisole could reversibly inhibit acetylcholinesterase in vivo and
prevent or delay the irreversible inhibition of this enzyme by dichlorvos. Such
a protective mechanism is found between physostigmine and organophosphates
(SOMANI and DUBE, 1989). One difficulty in accepting this hypothesis is that
levamisole did not significantly inhibit EChE in the rabbits of the present
study or in pigs (HSU, 1981a). But, levamisole pre-treatment significantly
increased the inhibitory effect of dichlorvos on EChE, suggesting that
levamisole and dichlorvos may bind at different sites of the enzyme.
Furthermore, the calculated percentages of EChE inhibition suggest that
levamisole pre-treatment did not potentiate the anticholinasterase action of
dichlorvos in rabbits. Also, in vitro inhibition of rabbit plasma and
erythrocyte cholinesterase by a combination of levamisole and dichlorvos did not
exceed the sum of inhibitions induced by either drug alone (ALDABAGH and
MOHAMMAD, 1998). The findings of the present study with regard to cholinesterase
inhibition correlate with those reported in pigs treated with dichlorvos and
levamisole (HSU, 1981a).

In conclusion, adverse drug interaction
is not likely to occur in animals concurrently treated with levamisole and
dichlorvos; instead, levamisole pre-treatment may diminish the toxic effects of
dichlorvos.

Acknowledgement
This study was
supported by the College of Veterinary Medicine, University of Mosul.

References

ALDABAGH, I. I., F. K. MOHAMMAD (1998):
In vitro inhibition of rabbit blood cholinesterase by levamisole and dichlorvos.
Iraqi J. Vet. Sci. 11, 17-22.

ANONIMOUS (1986): Organophosphorus
Insecticides: A general Introduction. World Health Organization. Geneva. pp.
1-100.

BRUNING, J. L., B. L. KINTZ (1977):
Computational Handbook of Statistics. Scott, Foresman and Co. Glenview, IL. p.
18.

DALVI, R. R. (1990): Pharmacology and
toxicology of levamisole in domestic animals: a review. Int. J. Anim. Sci. 5,
15-19.

EYRE, P. (1970): Some pharmacodynamic
effects of nematocides: methyridine, tetramisol and pyrantel. J. Pharm.
Pharmacol. 22, 26-36.

FARIS, G. A-M., F. K. MOHAMMAD (1997):
Prevention and treatment of dichlorvos-induced toxicosis in mice by
diphenhydramine. Vet. Hum. Toxicol. 39, 22-25.

FIKES, J. D. (1990): Organophosphorus
and carbamate insecticides. Vet. Clin. N. Am: Small Anim. Prac. 20, 353-367.

FORSYTH, B. A. (1966): Tetramisole: A
new athelmintic for sheep. Aust. Vet. J. 42, 412-419.

HSU, W. H. (1980): Toxicity and drug
interaction of levamisole. J. Am. Vet. Med. Assoc. 176, 1166-1169.

HSU, W. H. (1981a): Drug interactions
of levamisole with pyrantel tartarate and dichlorvos in pigs. Am. J. Vet. Res.
42, 1912-1914.

HSU, W. H. (1981b): Toxic effects of
levamisole and dichlorvos on ventilatory flow, phrenic nerve activity and
diaphragm movement in anesthetized dogs. The 62nd Annual Meeting of
the Conference of research Workers in Animal Disease. Chicago, IL. Abstract No.
34.

MARRINER, S., J. ARMOUR (1986):
Nematode infections of domestic animals, gastrointestinal infections. In:
Chemotherapy of Parasitic Diseases. (Campbell, W.C., R.S. Rew, Eds.). Plenum
Press. New york. pp. 297-299.

MOHAMMAD, F. K., V. E. V. ST. OMER
(1982): Modifications of Michel's electrometric method for rapid measurement of
blood cholinesterase activity in animals: a minireview. Vet. Hum. Toxicol. 24,
119-121.

PHILIP, R. D., D. STONE (1967):
Anthelmintic and toxicity studies with tetramisole. II. Toxicity studies in
sheep and goats. J. South Afr. Vet. Med. Assoc. 38, 287-293.

RUNYON, R. P. (1977): Nonparametric
Statistics. A Contemporary Approach. Addison Wesley Publishing Co., Reading,
Massachusetts. pp. 42-44.

SOMANI, S. M., S. N DUBE (1989):
Physostigmine an overview as a pretreatment drug for organophosphate
intoxication. Int. J. Clin. Pharmacol. Therap. Toxicol. 27, 367-387.

Received: 13 August 1998
Accepted:
15 January 1999

ALDABAGH, I. I., F. K. MOHAMMAD:
Smanjivanje toksicnosti diklorvosa u kunica levamisolom. Vet. arhiv 69, 29-37,
1999.

SAZETAK

Istrazivana je toksicnost diklorfosa i
interakcije s levamisolom na muskim kunicima. Znakovi otrovanja diklorvosom (25,
50 i 75 mg/kg, potkozno) u kunica ovisili su o dozi, a najvisa doza je u svih
uzrokovale pojacano slinjenje, suzenje, ataksiju i drhtanje. Ta doza uzrokovala
je i konvulzije u 50% i smrt u 75% kunica. Levamisol na 25% i 75% nije znacajno
utjecao na aktivnost acetilkolinesteraze eritrocita (EChE) 2 i 24 sata nakon
trovanja. Oralne doze diklorvosa od 10 i 20 mg/kg izazvale su, ovisno o dozi,
znakove kolinergicne toksicnosti. Doza od 20 mg/kg diklorvosa izazvala je smrt
75% kunica. Prethodno davanje levamisola u dozi od 50 mg/kg zastitilo je kunice
od otrovanja diklorvosom (20 mg/kg), odnosno sprjecilo je pojavu konvulzija,
dahtanja i smrti, a znacajno je povecalo latenciju pocetka drhtanja u usporedbi
s kontrolnom skupinom tretiranom diklorvosom. Kombinirani tretman levamisolom
(50 mg/kg) i diklorvosom (10 mg/kg) izazvao je znacajno visi stupanj EChE
inhibicije na 2 i 24 sata nakon davanja u usporedbi sa skupinom koja je dobila
samo diklorvos. Podaci upucuju na zakljucak da levamisol stiti kunice otrovane
diklorvosom, neovisno o inhibiciji EChE.

Kljucne rijeci: levamisol,
diklorvos, kunic, simptomi kolinesteraze eritrocita, organofosfati

Back