Veterinarski arhiv 69 1

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VETERINARSKI ARHIV = 69 (1),=20 29-37, 1999

ISSN 1331-8055 = Online
ISSN=20 0372-5480 Printed in Croatia

Reduction of = dichlorvos-induced=20 toxicosis in
rabbits by levamisole

Imad Ibrahim = Aldabagh and Fouad=20 Kasim Mohammad*

Department of = Physiology,=20 Biochemistry and Pharmacology,
College of Veterinary Medicine, = University of=20 Mosul, Iraq

* Contact = address:
Prof. Dr. Fouad=20 Kasim Mohammad,
Department of Physiology, Biochemistry and = Pharmacology,=20 College of Veterinary Medicine, University of Mosul, P.O. Box 11136, = Mosul, Iraq=20

ALDABAGH, I. I., F. K. = MOHAMMAD:=20 Reduction of dichlorvos-induced toxicosis in rabbits by levamisole. Vet. = arhiv=20 69, 29-37, 1999.

ABSTRACT =

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

Key words: = levamisole,=20 dichlorvos, rabbit, erythrocyte cholinesterase symptoms, organophosphate =

Introduction =

Levamisole, the 1-isomer = of=20 tetramisole, is an anthelmintic used in veterinary practice (MARRINER = and=20 ARMOUR, 1986; DALVI, 1990). Levamisole poisoning in animals is = characterised by=20 salivation, lacrimation, miosis, hyperventilation or dyspnea, frequent = urination=20 and defecation, ataxia, tremors and convulsion before death (HSU, 1980, = 1981a,=20 1981b; DALVI, 1990). These signs of levamisole poisoning resemble those = of=20 organophosphorus (OP) insecticides which cause muscarinic, nicotinic and = central=20 nervous system effects (ANON., 1986; FIKES, 1990). Thus, it appears that = there=20 is the possibility of potentiated toxic effects when levamisol and OP=20 insecticides are concurrently used in animals.

Treatment of mice (HSU, = 1980) and pig=20 (HSU, 1981a) with the PO insecticide dichlorvos did not increase the = toxicity of=20 levamisole. Similarly, the PO insecticide diazinon did not increase the = toxicity=20 of tetramisole in sheep (FORSYTH, 1966). However, in these studies the=20 insecticides were given prior to levamisole (HSU, 1980, 1981a) or = tetramisole=20 (FORSYTH, 1966). The present study was undertaken in rabbits in order to = further=20 examine levamisole-induced toxicosis and its interaction with = dichlorvos, taking=20 account of the administration of levamisole before dichlorvos dosing. = This=20 approach provided some new information about the ability of levamisole = in=20 reducing the toxic effects of dichlorvos.

Materials and methods=20

Male cross-bred rabbits = with masses of=20 1.2-1.9 kg were used. They were housed at a temperature of 22=B12 =B0C = with 10/14 h=20 light/dark cycle. The animals had free access to food and water. =

Experiment I, toxic = effects of=20 levamisole:

Rabbits (n=3D4/group) = were treated=20 subcutaneously (s.c.) with levamisole hydrochloride (7.5%, Phenex, = Belgium) at=20 dose rates of 25, 50 and 75 mg/kg body mass. The control group received=20 physiological saline solution at 1 ml/kg, s.c. Levamisole doses = were=20 determined in a preliminary experiment in rabbits. After levamisole = injection,=20 each rabbit was observed until death, or for 2 h, for the appearance of = signs of=20 poisoning which included salivation, lacrimation, frequent urination and = defecation, ataxia, tremor and convulsion.

Two and 24 h after = levamisole=20 administration (25 and 50 mg/kg, s.c.) blood samples were obtained from = the ear=20 vein by a heparinised syringe. Erythrocytes were separated by = centrifugation of=20 the blood samples at 3000 rpm for 15 minutes. An aliquot of 0.1 ml of=20 erythrocytes was used for the determination of erythrocyte = acetylcholinesterase=20 (EChE) activity by the modified electrometric method described = previously=20 (MOHAMMAD and OMER, 1982).

Experiment II, effects = of levamisole=20 pre-treatment on dichlorvos-induced toxicosis:

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

The latencies to onset of = tremor and=20 death within 2 h were recorded. The activity of EChE was determined as = mentioned=20 in experiment I prior to drug administration (time 0, baseline) and = thereafter 2=20 and 24 h after dichlorvos dosing at 10 mg/kg. The percent of EChE = inhibition was=20 determined as follows:

% inhibition=3D SEChE activity (baseline)-EChE activity = at 2 or 24=20 h/EChE activity (baseline)C =D7 = 100=20

In both experiments, the = rabbits were=20 retained for 3 days for any delayed death.

Statistics: Frequency = data were=20 subjected to Fisher's exact probability test (RUNYON, 1977). The = severity score=20 to toxicity was analysed by Mann-Whitney-U-test (RUNYON, 1977). Tremor = times=20 were compared by Unpaired Student's-t-test (BRUNING and KINTZ, 1977). = The level=20 of significance was at P<0.05.

Results =

Subcutaneous injection of = levamisole in=20 rabbits induced signs of poisoning that were dose dependent (Table 1). = At the=20 highest dose (75 mg/kg) tested, levamisole significantly increased the=20 occurrence of excessive salivation, lacrimation, ataxia and tremor by = 100%, and=20 caused convulsion and death in 50 and 75% of the rabbits, respectively = (Table=20 1). The mean=B1SE latency to onset of death was 55=B120 minutes. The 50 = mg/kg dose=20 of levamisole was well tolerated by the rabbits which showed salivation = (75%),=20 lacrimation (50%) as well as ataxia and tremor (25%) in comparison with = the=20 control group (Table 1). Levamisole at 25 and 50 mg/kg did not = significantly=20 affect EChE activity at 2 and 24 h after treatment (Table 2). However, = in the=20 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=20 in rabbits

Variable (% = occurrence)=20	Control =	Levamisole = (mg/kg, s.c.)=20
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=20

N=3D4 rabbits/dose group. = Each rabbit was=20 observed until death or for 2 h after levamisole = treatment.
*Significantly=20 different from the corresponding control value, P<0.05.

Table 2. Erythrocyte=20 acetylcholinesterase activity in rabbits treated with levamisole =

Levamisole (mg/kg, s.c.)=20	Difference pH / = 30 minutes=20
2 h =	24 h =
0 (saline) =	0.45=B10.02 =	0.39=B10.04 =
25	0.37=B10.03 =	0.39=B10.06 =
50	0.38=B10.05 =	0.37=B10.06=20

Dichlorvos dosing at 10 = or 20 mg/kg in=20 rabbits dose-dependently induced signs of toxicosis characteristics of=20 cholinesterase inhibition and subsequent cholinergic overstimulation = (Table 3).=20 These signs were salivation, lacrimation, frequent urination and = defecation,=20 tremor, ataxia, muscle fasciculation, flat body appearance, gasping and=20 convulsion (Table 3). The 20 mg/kg dose of dichlorvos caused death in 3 = out of 4=20 rabbits. The latency to onset of death was 25=B17 minutes.

Table 3. Effect of = levamisole on=20 dichlorvos-induced toxicosis in rabbits

Variable (% = occurrence)=20	Dichlorvos = (mg/kg, orally)=20
10 =	20 =
Levamisole = (mg/kg, s.c.)=20
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=20	100 =	25 =	25 =	100 =	50 =
Flat body = appearance=20	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*=20	15 =	11* =	8* =	28 =	10.5*=20

Rabbits (4/group) were = pretreated with=20 levamisole 30 minutes before dichlorvos dosing. Each rabbit was observed = until=20 death or for 2 h after dichlorvos dosing. *The severity score of = toxicity for=20 each rabbit was the summation of grades of sign of toxicosis from 0 to = 7:=20 0=3Dnormal; 1=3Dhyperactivity and/or agitation; 2=3Dsalivation; = 3=3Dtremor; 4=3Dmuscle=20 fasciculation; 5=3Dflat body appearance; 6=3Dconvulsion; 7=3Ddeath. For = reference, the=20 highest severity score when all the signs of toxicosis mentioned above = are=20 observed in an individual rabbit would be 28. *Significantly different = from the=20 respective control (0 dose) value, P<0.05.

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

Table 4. Effect of = levamisole on the=20 latency to onset of tremors in rabbits dosed with dichlorvos

Treatment (mg/kg) =	Latency to onset = of tremor=20 (min)
Saline + Dichlorvos = (10)=20 (control)	8.0 =B1 1.2 =
Levamisole (25) + = Dichlorvos (10)=20	9.0 =B1 0.7 =
Levamisole (50) + = Dichlorvos (10)=20	11.8 =B1 1.3* =
Saline + Dichlorvos = (20)=20 (control)	3.8 =B1 0.5 =
Levamisole (50) + = Dichlorvos (20)=20	8.8 =B1 1.0*=20

The values are mean=B1SE = of 4=20 rabbits/group. Levamisole was injected subcutaneously 30 minutes before = oral=20 dosing of dichlorvos. *Significantly different from the respective = control=20 value, P<0.05.

Fig. 1. Percentage of = erythrocyte=20 acetylcholinesterase (EChE) inhibition in rabbits dosed orally with = dichlorvos=20 (10 mg/kg) after pre-treatment with saline (control) and levamisole at = 25 and 50=20 mg/kg, s.c.. Pre-treatment (baseline) values (mean=B1SE) of EChE = activity of the=20 three groups were 0.44=B10.02, 0.38=B10.03 and 0.41=B10.02, difference = pH/30 minutes,=20 respectively. %inhibition=3DSEChE = activity=20 (baseline)-EChE activity at 2 or 24 h / EChE activity (baseline)C =D7 100. Levamisole was injected 30 = minutes before=20 dichlorvos dosing. *Significantly different from the control = (dichlorvos-saline)=20 group, P<0.05.

Combined levamisole (50 = mg/kg) and=20 dichlorvos (10 mg/kg) treatments caused a significantly higher degree of = EChE=20 inhibition at 2 and 24 h after dichlorvos dosing in comparison with the=20 dichlorvos-treated group (Figure 1).

Discussion =

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

The mechanism of the = protective effect=20 of levamisole against dichlorvos-induced toxicosis is not clear at = present.=20 Tetramisole (EYRE, 1970) and levamisole (ALDABAGH and MOHAMMAD, 1998) = were found=20 to inhibit acetylcholinesterase activity in vitro. Considering the fact = that=20 levamisole at 25 and 50 mg/kg decreased (though non-significantly) EChE = in the=20 rabbits by 18 and 16%, respectively (Table 2), it is therefore possible = that=20 levamisole could reversibly inhibit acetylcholinesterase in vivo = and=20 prevent or delay the irreversible inhibition of this enzyme by = dichlorvos. Such=20 a protective mechanism is found between physostigmine and = organophosphates=20 (SOMANI and DUBE, 1989). One difficulty in accepting this hypothesis is = that=20 levamisole did not significantly inhibit EChE in the rabbits of the = present=20 study or in pigs (HSU, 1981a). But, levamisole pre-treatment = significantly=20 increased the inhibitory effect of dichlorvos on EChE, suggesting that=20 levamisole and dichlorvos may bind at different sites of the enzyme.=20 Furthermore, the calculated percentages of EChE inhibition suggest that=20 levamisole pre-treatment did not potentiate the anticholinasterase = action of=20 dichlorvos in rabbits. Also, in vitro inhibition of rabbit plasma = and=20 erythrocyte cholinesterase by a combination of levamisole and dichlorvos = did not=20 exceed the sum of inhibitions induced by either drug alone (ALDABAGH and = MOHAMMAD, 1998). The findings of the present study with regard to = cholinesterase=20 inhibition correlate with those reported in pigs treated with dichlorvos = and=20 levamisole (HSU, 1981a).

In conclusion, adverse = drug interaction=20 is not likely to occur in animals concurrently treated with levamisole = and=20 dichlorvos; instead, levamisole pre-treatment may diminish the toxic = effects of=20 dichlorvos.

Acknowledgement
This = study was=20 supported by the College of Veterinary Medicine, University of Mosul.=20

References =

ALDABAGH, I. I., F. K. = MOHAMMAD (1998):=20 In vitro inhibition of rabbit blood cholinesterase by levamisole and = dichlorvos.=20 Iraqi J. Vet. Sci. 11, 17-22.

ANONIMOUS (1986): = Organophosphorus=20 Insecticides: A general Introduction. World Health Organization. Geneva. = pp.=20 1-100.

BRUNING, J. L., B. L. = KINTZ (1977):=20 Computational Handbook of Statistics. Scott, Foresman and Co. Glenview, = IL. p.=20 18.

DALVI, R. R. (1990): = Pharmacology and=20 toxicology of levamisole in domestic animals: a review. Int. J. Anim. = Sci. 5,=20 15-19.

EYRE, P. (1970): Some = pharmacodynamic=20 effects of nematocides: methyridine, tetramisol and pyrantel. J. Pharm.=20 Pharmacol. 22, 26-36.

FARIS, G. A-M., F. K. = MOHAMMAD (1997):=20 Prevention and treatment of dichlorvos-induced toxicosis in mice by=20 diphenhydramine. Vet. Hum. Toxicol. 39, 22-25.

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

FORSYTH, B. A. (1966): = Tetramisole: A=20 new athelmintic for sheep. Aust. Vet. J. 42, 412-419.

HSU, W. H. (1980): = Toxicity and drug=20 interaction of levamisole. J. Am. Vet. Med. Assoc. 176, 1166-1169. =

HSU, W. H. (1981a): Drug = interactions=20 of levamisole with pyrantel tartarate and dichlorvos in pigs. Am. J. = Vet. Res.=20 42, 1912-1914.

HSU, W. H. (1981b): Toxic = effects of=20 levamisole and dichlorvos on ventilatory flow, phrenic nerve activity = and=20 diaphragm movement in anesthetized dogs. The 62^nd Annual = Meeting of=20 the Conference of research Workers in Animal Disease. Chicago, IL. = Abstract No.=20 34.

MARRINER, S., J. ARMOUR = (1986):=20 Nematode infections of domestic animals, gastrointestinal infections. = In:=20 Chemotherapy of Parasitic Diseases. (Campbell, W.C., R.S. Rew, Eds.). = Plenum=20 Press. New york. pp. 297-299.

MOHAMMAD, F. K., V. E. V. = ST. OMER=20 (1982): Modifications of Michel's electrometric method for rapid = measurement of=20 blood cholinesterase activity in animals: a minireview. Vet. Hum. = Toxicol. 24,=20 119-121.

PHILIP, R. D., D. STONE = (1967):=20 Anthelmintic and toxicity studies with tetramisole. II. Toxicity studies = in=20 sheep and goats. J. South Afr. Vet. Med. Assoc. 38, 287-293.

RUNYON, R. P. (1977): = Nonparametric=20 Statistics. A Contemporary Approach. Addison Wesley Publishing Co., = Reading,=20 Massachusetts. pp. 42-44.

SOMANI, S. M., S. N DUBE = (1989):=20 Physostigmine an overview as a pretreatment drug for organophosphate=20 intoxication. Int. J. Clin. Pharmacol. Therap. Toxicol. 27, 367-387. =

Received: 13 August = 1998
Accepted:=20 15 January 1999

ALDABAGH, I. I., F. K. = MOHAMMAD:=20 Smanjivanje toksicnosti diklorvosa u kunica levamisolom. Vet. arhiv 69, = 29-37,=20 1999.

SAZETAK =

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

Kljucne rijeci: = levamisol,=20 diklorvos, kunic, simptomi kolinesteraze eritrocita, organofosfati =