Investigations on the Efficacy of Some Anticestode Compounds against Echinococcus granulosus in the Dog *

Dr. Tibor KASSAI — Dr. Péter R E D L — Csilla TAKÁTS — Dr. Ferenc HOLLÓ — Dr. Károly MAGYAR — Dr. János G Y E N E S Helminthological Reserch Laboratory, Department of Parasitology, Veterinary University, Budapest — P H Y L A X I A Veterinary Biologicals and Feedstuffs Ltd., Budapest

"Investigations on the efficacy of some anticestode compounds against Echinococ­cus granulosus in the dog" - Kassai , T. - Redl, P. - T a k á t s , Cs, - Holló, F . - Magyar, K. - Gyenes, J. - Paras i t . Hung. 8. 29-35. 1975.

ABSTRACT. The efficiency of single treatments w i th bunamidine hydroxynaphthoate (BHN, Burroughs Wellcome and Co. ), bunamidine hydrochlor ide (BHC1, "Sco-laban" Burroughs Wellcome and Co. ), niclosamide (NA, "Dayermin" Chinoin) and cantrodifene (CF, "Lopatol" , CIBA-GEIGY) against 6-week old E . ganulosus was tested on 113 exper imental ly infected dogs'. BHN at 100 mg /kg removed 95.1-97.2 per cent of the worms f rom 89 per cent of the treated dogs. FoUowing treatment with NA or CF, considerable w o r m reduction was recorded in 28 and 14 per cent of the animals, respectively, while results of the BHCl- t rea tment were inconclu­sive. BHN i n biscui t formulat ion was found to f u l f i l l p rac t ica l requirements of control schemes involving regular dosing.

Despite many previous research endavours to acquire an appropriate chemotherapeu-t ic agent against Echinococcus granulosus, - treatment of canine echinococcosis remains a p rob lem deserving of attention, for even those few compounds that have been recom­mended against this parasite have been indicted for inadequate efficiency or undesirable side effects.

Since 1965 data have accumulated on the anti Echinococcus ac t iv i ty of several naph­thalene derivat ives, of which bunamidine (N : N - d i - n - butyl-4 -hexyloxy-1 - naphthamidine) hydrochlor . ide x has become widely known on account of i ts re l iab le efficiency ( B A L T Z L Y e t a l . , 1965; FORBES, *966; ZUKOAflC and WIKERHAUSER, 1967; G E M M E L and SHEA­RER, 1968; BLOOD et a l . , 1968; DEMIDOV et a l . , 1974; AZIMOV é s AMINZHANOV, 1974). However, l abora tory and f ie ld studies, conducted in New Zealand, revealed toxic, sometimes fatal complications after t reatment wi th bunamidine hydrochloride (THOMSON, 1973; MENRATH et a l . , 1973).

In labora tory t r i a l s a water- insoluble salt of bunamidine, bunamidine hydroxynaphthoate, also proved very efficient, and less toxic even at higher and repeated doses (SHEARER and G E M M E L , 1969; NIKI T I N et a l . , 1973; G E M M E L and OUDEMANS, 1974/a).

Niclosamide ( N - / - 2 - c h l o r - 4 - n i t r o p h e n y l / - 5 - c h l o r - s a l i c y l - a m i d e ) is known to be highly effective against common tapeworms of the dog. However, confl ict ing resul ts have been repor ted about i t s action against E. granulosus (KURELEC and RIJAVEC, 196.1; FOR­BES, 1963; D E L A K e t a l . , 1963; BABOS et a l . , 1964).

Recently a broad spectrum anthelmintic, cantrodifene (4- i so th iocyano-4*-n i t ro-d iphe-ny l e t h e r ) x x has also been claime.d to be highly effective against E. granulosus in the dog (BORAY, von O R E L L I and SARAS I N , 1974; G E M M E L and OUDEMANS, 1974/b).

* Presented at the Second European Mul t icol loquy of Parasi tology, 1-6 September, 1975, T r o g i r , Yugoslavia. X "Scolaban", Burroughs Wellcome and Co. X X " L o p a t o l " , CIBA-GEIGY

This paper reports the resul ts obtained by a single high dose leve l t reatment wi th buna­mid ine hydroxynaphthoate against E . granulosus as w e l l as experiences wi th a medicated b iscui t formulat ion developed i n order to meet the requirements of an easy application. L i m i t e d comparative observations on the efficacy of bunamidine hydrochlor ide , n i c lo s ­amide and cantrodifene to this parasi te have also been made.

Material and Methods

LABORATORY EXPERIMENTS

Anima l s . Dogs of both sexes and mixed breed over 4 months of age were used. Each was vaccinated against canine distemper and then treated by "Devermin" (Chinoin) to e l i ­minate common tapeworms or ig ina t ing f r o m natura l infect ion.

Infective ma te r i a l . F o r the exper imenta l infection scolices were col lected f rom pig l i v e r s containing hydatid cysts. Pooled hydatid f lu id was passed through a syringe seve­r a l t imes to obtain free indiv idual scolices in the suspension. A di lut ion method was used to determine the concentration of viable scolices. Infective m a t e r i a l was kept at 4 °C over­night and used for infect ion wi th in 30 hours of slaughter of the pigs.

Exper imenta l detai ls . Three experiments were conducted involving a to ta l of 113 dogs. Animals were o ra l l y infected w i t h 30 000 (Exp, 2) or 50 000 (Exps. 1 and 3) scolices each, adminis tered by syr inge i n 20 m l of f lu id . A l l exposed animals developed infection wi th E . granulosus. On day 41 or 42 after infection a control group was allocated for each ex­per iment (Groups D, H and K ) . The remainder were divided into groups, weighed and t rea ted as indicated in Table 1. Results were assessed between days 3 and 19 after t r ea t ­ment by post m o r t e m determinat ion of the individual w o r m counts. Fo l lowing about 20 hours of starvation dogs were k i l l e d , each s m a l l intestine removed, and divided into four par ts of equal length. Each par t was opened, the content careful ly collected in a P e t r i d ish , and mixed wi th the mucosal and submucosal layers of the intest inal segment stripped off gently with a knife. This m a t e r i a l was examined in sma l l quantities between compressed glassplates under dissect ing microscope. A l l scolices were counted. In some heavily i n ­fected animals (Exp. 3) an aliquot sample was mic roscop ica l ly examined and the to ta l number of scolices calculated on a weight basis. In a l l cases worm counts were made w i t h i n 24 hours of autopsy.

C r i t e r i a for assessment. I n most groups animals both wi th low and high w o r m counts were found rendering calculat ion of group mean values ra ther pointless. Each control dog - except one an imal - had a w o r m burden wel l in excess of 500 worms , the mean w o r m counts being 3370, 2447 and 13 225 in Groups D, H and K, respect ively. There­fore , dogs found to harbour more than 500 scolices were classif ied as non recovered (NR), and those wi th fewer worms as recovered (R). Thus, anthelmintic efficiency is expressed by the combination of two parameters : by the propor t ion of dogs recovered after treatment and by the per cent reduction of the w o r m count of the recovered dogs as compared to the mean w o r m burden of the controls (Table 2).

Drugs used

1. ) Bunamidine hydroxynaphthoate powder (Burroughs Wellcome and Co. )

1x75 mg /kg in a 10% suspension wi th Carbowax 400 - Group A 1x100 m g / k g in a 10/o suspension wi th Carbowax 400 - Group B 1x100 m g / k g i n biscui t formula t ion - Group E 1x100 m g / k g mixed wi th minced raw meat (in the food) - Group F

F i g . 1. Exper imenta l medicated biscui ts containing 1 g bunamidine hydroxynaphtoate each (for 10 kg body weight)

Table 1 Exper imenta l details and w o r m recoveries

Group Treatment N u m ­ber of dogs '

W o r m counts Group Treatment

N u m ­ber of dogs ' ind iv idua l mean

A BHN susp. 15 R X 203, 46, 22, 5, 4, 4, 0, 0 35 1x75 m g / k g NR 5766, 2884, 2311, 1446,

1124, 1047, 672 2178

B BHN susp. 15 R 393, 249, 201, 124, 85, 80 44, 42, 40, 16, 6, 0 99

m

1x100 m g / k g NR 1261, 737, 582 860

m NA tabl . 14 R 293, 288, 103, 96 195 m 1x500 m g / k g NR 9190, 6730, 5662, 4785, 4543, 2526, 2380, 1587, 1527, 586

3951

D Contro l 14 R 171 171 NR 7647, 5876, 5496, 4084,

4017, 3428, 2937, 2236, 2137, 21 13, 2053, 1125, 671

3370

E BHN biscui t 9 R 451, 359, 86, 63, 7, 6, 1, 0 121 1x100 m g / k g NR 715 715

F BHN i n the food 9 R 321, 161, 51, 18, 2, 0, 0, 0 69 1x100 m g / k g NR 3950 3950

CM G CF tabl . . 8 R 399, 382 390

Exp

. 1x200 m g / k g NR 9166, 8352, 6814, 6227 4415, 2679

6275

H Contro l 7 R - -NR 5290, 4321, 2995, 1892,

1636, 552, 546 2447

I CF tab l . 8 R _ -1x200 m g / k g NR 16970, 14670, 10980, 10885

9670, 6430, 3350, 2100 9382

CO J BHC1 t ab l . 5 R 77, 61 , 5 48 1x100 m g / k g NR 12693, 1735 7214

Contro l 9 R - -26000, 20860, 19060, 15040 10300, 8770, 7710, 6610 4670

13225

R • recovered NR = non recovered

2. ) Bunamidine hydrochloride tablets ("Scolaban" Burroughs Wellcome and Co. )

1x100 m g / k g - Group J

3. ) Niclosamide tablets {"Devermin" Chinoin) 1x500 m g / k g - Group C

4. } Cantrodifene tablets {"Lopatol" CIBA-GEIGY) 1x200 m g / k g - Groups G and I

F I E L D T R I A L

One of the greatest difficult ies concerning anthelmint ic medication of the dog is the need for res t ra in t . Vomi t ing occurs frequently i n stressed dogs i n t e r f e r i ng wi th the ef­f ic iency of the t rea tment . Hence, b iscui t fo rmula t ion was considered suitable for easy adminis t ra t ion . Biscu i t s (formulations A and B) containing 1 g (5%) of bunamidine hydro­xynaphthoate each were prepared w i t h Carbowax - 4000, carti lage pulp and "Protecan " ( F i g . 1), Fo rmu la t i on A was used i n Exp. 2 (Group E) . This was followed by a f ie ld t r i a l in which 122 and 35 dogs, a l l kept in the i r usual habitat, were given medicated biscui ts of A and B formulat ion, respect ively, i n o rde r to test the i r pala tabi l i ty .

Results

LABORATORY EXPERIMENTS

BHN given i n the food was read i ly consumed by dogs of Group F , but biscuits were p a r t i a l l y or to t a l ly refused by 5 of 9 dogs of Group E wi th in 2 and a half hour of obser­vation. Remaining biscui ts were then broken, mixed wi th minced raw meat and fed. A f ­te r treatments no untoward sequelae were noted except vomit ing of 2 dogs given BHN i n suspension (one each in Groups A and B),

Table 2

Summary of resul ts of t reatment t r i a l s against 6-week old E . granulosus

Treatment

P ropo r t i on of dogs recovered after t rea tment

Pe r cent reduc­t ion of w o r m burden in reco­vered dogs

Treatment number per cent

Pe r cent reduc­t ion of w o r m burden in reco­vered dogs

BHN susp, 75 m g / k g 8/15 53 99, 0 BHN susp, 100 m g / k g 12/15 80 97, 1 B H N i n the food 100 mg/kg 8/9 89 97, 2 BHN biscui t 100 m g / k g 8/9 89 95, 1 BHC1 tabl . 100 m g / k g 3/5 60 99, 6 NA tab l . 500 m g / k g 4/14 28 94, 3 CF tab l . 200 m g / k g 2/16 14 84, 1

W o r m recovery data are given in Table 1 and the resul ts are summar ized in Table 2. I t is c lear f rom the data that against 6-week old E . granulosus a single t reatment of BHN at a dose leve l of 100 mg /kg effected the greatest w o r m removal of the four compounds involved i n this study. When given i n the food or i n biscui t , BHN removed 95 to 97 per cent of worms in 89 per cent of t reated animals . Only r a r e l y was a worm- f r ee condi ­t i o n achieved.

Treatment with BHC1 gave inconclusive results in wiew of the great range of v a r i a ­t ion of worm counts seen in the 5 dogs of Group J.

NA at 500 m g / k g showed l i m i t e d efficiency resul t ing in substantial worm reduction in 4 (28 per cent) of the treated 14 dogs only (Group C).

S imi l a ry , l i t t l e ac t iv i ty against E. granulosus was shown in either Groups G and I dosed by 200 m g / k g of cantrodifene. (In Exps. 2 and 3 "Lopato l" tablets of different code numbers were used.) This drug proved active in 2 of 16 dogs (14 per cent) only.

F I E L D TRIALS

F r o m the data of Table 3 it is seen that biscuits containing BHN were readi ly consu­med by the non excited dogs. Previous starvation was apparently not needed. Nei ther vomi t ing nor other side effects were manifest wi th in 1 hour after t reatment . The 98,3 and 88, 5 per cent to ta l consumption of biscui t A and B, respectively, indicate that the pala tabi l i ty of formula t ion A is superior to that of B.

Table 3

Pa la tab i l i ty f ield t r i a l wi th BHN in biscui t formulat ion

Biscuit A Biscuit B

To ta l number of dogs 122 35 Dogs consuming to ta l dose 120 31

immedia te ly 115 30 wi th in 15 minutes 2 1 wi th in 1 hour 3 0

Dogs consuming a part of the dose 1 1 Dogs refusing consumption 1 3

Discussion

The few reports on the action of bunamidine hydroxynaphthoate against mature (8 to 9-week old) E. granulosus recommend 2 doses given at 2 or 4 days in te rva l in the food at a dose level of 12, 5 to 64 mg/kg (SHEARER and G E M M E L , 1969; G E M M E L and OUDEMANS, 1974/a). This course of t reatment may reduce worm burden by 99,8 per cent in the average. No increase of efficiency occurred when the level was raised to 100 m g / k g (SHEARER and G E M M E L , 1969).

Complete e l imina t ion of a l l Echinococcus is obtainable by additional treatments ra ther than by giving higher doses. It has been calculated that in order to achieve a "one h i t " absolute k i l l of worms , an inapplicable rate exceeding 400 m g / k g may be needed (GEM­M E L and OUDEMANS, 1974/a). Against immature , 3 to 4-week old worms a 91 per cent reduction has been shown by a 2x50 mg /kg treatment (SHEARER and G E M M E L , 1969) and against 5-week old worms a 100 per cent efficacy of the same dose leve l has been reported (NIKITIN et a l . , 1973). It has also been observed that untoward sequelae such as vomi t ing and diarrhoea occur more frequently fol lowing the second rather than the f i r s t t reatment (SHEARER and G E M M E L , 1969).

This study attempted to define the efficacy of a single high dose leve l treatment wi th bunamidine hydroxynaphthoate and to test a formulat ion which may facil i tate massive application of the drug in control schemes.

The results obtained demonstrate that a single treatment at 100 mg /kg is highly effec­t ive against 6-week old worms , though normal ly is not expected to cause e l iminat ion o f

a l l worms . An absolute k i l l of the Echinococcus burden would necessitate several sub­sequent t reatments, the cost of which is not commensurate wi th the anticipated resul ts .

Dogs readi ly accept this dose i f i t is del ivered in a biscuit formula t ion thus avoiding the need for forcefeeding. Also t reatment wi th the medicated biscuit can be c a r r i e d out by the owner and i f necessary, can easily be repeated. Undesirable side effects of the t reatment are m i n i m a l and t ransient .

Our observation on the inadequate efficiency of niclosamide at 500 m g / k g against E. granulosus is i n l i ne with the resul ts reported by KURELEC and RIJAVEC (1961), FOR^~ BES (1963) and D E L A K et a l . (1963). Therefore con t ra ry to the findings of BABOS et a l . (1964), niclosamide cannot be recommended for the treatment of canine echinococcosis.

Cantrodifene ("Lopatol") , given ei ther direct o r mixed wi th the food, has been p r o ­posed against E. granulosus at 200 m g / k g body weight once or twice wi th an in t e rva l of 24-48 hours. A single dose produced 92,6 to 99,8 per cent worm reduction (BORAY et a l . , 1974; G E M M E L and OUDEMANS, 1974b).In Exps. 2 and 3 of the present study, how­ever, a single t reatment wi th cantrodifene at 200 m g / k g un i fo rmly fai led to show sa t i s ­fac tory ac t iv i ty against E . granulosus.

In conclusion, of the compounds applied in the experiments described here bunami­dine hydroxynaphthoate proved to be the drug of choice for the t reatment of canine echi ­nococcosis. A single dose of 100 m g / k g was of re l iab le efficacy. Dogs readi ly accepted biscui ts contaning the drug at a concentration of 5 per cent, thus this formula t ion ap­pears to be we l l suited for wide scale regular dosing programmes.

KASSAI, T. — R E D L , P. — TAKÁTS, Cs. — HOLLÓ, F . — MAGYAR, K . — G Y E N E S , J . : Vizsgálatok néhány galandféreg ellenes szer hatékonyságáról Echinococcus granulosusszal fertőzött kutyákban

Ö s s z e s e n 113 k í s é r l e t i l e g f e r t ő z ö t t kutyán v i z s g á l t á k a köve tkező szerek egyszeri a-dagjainak Echinococcus -ellenes h a t é k o n y s á g á t : b u n a m i d i n - h i d r o x i n a f t o á t (BHN, Burroughs Wellcome and Co. ), bunamid in-h idrok lor id (BHC1, "Scolaban" Burroughs Wellcome and Co. ), n ik lozamid (NA, "Devermin" Chinoin) és a kantrodifen (KF, "Lopa to l " CIBA-GEI ­GY) . Legjobb e r e d m é n y t B H N - k e z e l é s s e l é r t e k e l . E szer 100 mg/kg-os adagja 9 5 , 1 -97, 2%-kal c s ö k k e n t e t t e a f é r e g s z á m o t a kezelt ebek 897o-ában. N A - d a l é s K F - n e l végze t t egyszeri k e z e l é s u tán a kutyák 28, i l l e tve 14%-ában köve tkeze t t be j e l e n t ő s f é r e g s z á m ­c s ö k k e n é s . A B H C l - k e z e l é s e r e d m é n y é b ő l nem lehetett k ö v e t k e z t e t é s t levonni . A kutyák s z í v e s e n e l fogyasz t j ák az 5%- BHN-ot t a r t a l m a z ó g y ó g y s z e r e s pá l c iká t , e z é r t nincs s z ü k ­s é g e r ő s z a k o s m e g f é k e z é s ü k r e . Ez a fo rmu lác ió alkalmasnak l á t s z i k az Echinococcus-ellenes akciók s o r á n s z ü k s é g e s s é vá ló rendszeres é s t ö m e g e s k e z e l é s e k m e g k ö n n y i t é s é -r e .

References

A Z I M O V , S.A. - AMINZHANOV, M . (1974): Effektivnost arekolina i bunamidina h i d r o -k lo r ida p r i echinokokkoze. - Ve te r ina r i a (Moskow), (10. ) 81-82.

BABOS, S. - KÖRMÖCZY, Gy. - L E H O C Z K I , Z. (1964): Ö s s z e h a s o n l í t ó v i z s g á l a t o k a Devermin A (Chinoin) és a Yomesan (Bayer) ebek g a l a n d f é r g e s s é g e el leni h a t á s á ­r ó l , kü lönös tekintet tel az echinococcosisra. - Magy. Ao. Lapja, J_9. 472-474.

B A L T Z L Y , R. - BURROWS, R. B. - HARFENIST, M . - F U L L E R , K. A. - K E E L I N G , J . E . D . - STANDEN, O. D. - HATTON, C. J. - NUNNS, V . J . - RAWES, D. A. -BLOOD, B . D . - MOYA, V. - L E L I J V E L D , J. L . (1965): A series of compounds active against cestodes. - Nature , 206. 408-409.

BLOOD, B . D . - MOYA, V. - L E L I J V E L D , J. L . (1968): Evaluation of selected drugs for the t reatment of canine echinococcosis. - B u l l . Wld. Hl th . O r g . , 39. 67-72.

BORAY, J. - von O R E L L I , M . - SARASIN, G. (1974): LOPATOL/ ' - cantrodifene a broad spectrum anthelmintic for dogs. - Manuscript of paper submitted at the 3rd Int . Congr. of Parasi tology, Munich.

DEMIDOV, N . V . - MATCHANOV, N . M . - AMINZHANOV, M . (1974): Usoversenstvova-nie metoda degelmint izaci i sobak p r i echinokokkoze. - Veter inar ia (Moskow), (10.) 80 -81 .

D E L A K , M . - KODRNJA, E. - RICHTER, S. - V R A Z I C , O. (1963): Is t razivanje djelat-nost i N - ( 2 ' - k l o r - 4 ' - n i t r o f e n i l ) - 5 - k l o r s a l i c i l a m i d a na trakovicavost pasc uzrokova-nu s Echinococcus granulosus. - Vet. A r h . (Zagreb), 33. 57-62.

FORBES, L . S . (1963): The efficiency of N - ( 2 ' - c h l o r - 4 ' - n i t r o p h e n y l ) - 5 - c h l o r sa l i cy lamid against Taenia hydatigena and Echinococcus granulosus infections in dogs.- Vet. R e e , 75. 321-324.

FORBES, L . S . (1966): The efficiency of bunamidine hydrochloride against young E c h i ­nococcus granulosus infection in dogs. - Vet. R e e , 79. 306-307.

G E M M E L , M . A. - OUDEMANS, G. C. (1974a): Treatment of Echinococcus granulosus and Taenia hydatigena i n dogs wi th bunamidine hydroxynaphthoate in a prepared food. - Res. Vet. Sei . , 16. 85-88.

G E M M E L , M . A. - OUDEMANS, G. C. (1974b): An evaluation of cantrodifene against E -chinococcus granulosus and Taenia hydatigena - c i t . : BORAY et a l . 1974.

G E M M E L , M . A . - SHEARER, G. C. (1968): Bunamidine hydrochlor ide. I ts efficiency against Echinococcus granulosus. - Vet. Ree, , 82. 252-256.

KURELEC, B , - RIJAVEC, M , (1961): O mogucnosti lecenja t rakovicavost i pasa i koko-s i Yomesanon. - Vet. g lasn . , 15. 602-606.

MENRATH, R, L , E, - SHARARD, A. - GRAY, K. W. - CAMERON, C. W. (1973): T o x i ­c i ty of bunamidine. 2. Metabolic effects. - N . Z. Vet. J . , 21. 212-215.

N I K I T I N , V . F . - RAMASANOV, V . T . - KASPAKBAEV, A . S. (1973): Effektivnost buna-midina oxinaphthoata p r i e x p e r i m e n t á l n o m echinococcose i multiceptose sobak. -BuU. Inst . Gelmint . imeni K . I . Skrjabina, _1_2. 55-57.

R E D L , P. - KASSAI, T. - TAKATS, Cs. - HOLLO, F . - MAGYAR, K. - GYENES, J. (1975): A bunamid inh id rox ina f toá t é s a n ik lozamid Echinococcus granulosus e l l e ­nes h a t é k o n y s á g á n a k v i z s g á l a t a ku tyákban . - Magy. Ao. Lapja, 30. 332-335.

SHEARER, G. C. - G E M M E L , M . A. (1969): The efficiency of bunamidine hydroxynaph­thoate against Echinococcus granulosus in dogs. - Res. Vet. Se i . , 10. 296-299.

THOMSON, G. A. (1973): The cont ro l of canine cestode infection in New Zealand. - Proc . 6th Symposium of the W. A. A. V. P. Vienna.

ZUKOVIC, M . - WIKERHAUSER, T. (1967): Is t raz ivanja anthelmintickog djelovanja Sco-labana na t rakavicu E. granulosus u psa. - Vet. A r h . (Zagreb), 37. 163-166.

Received: 10 June, 1975 Dr . KASSAI, T. T A K A T S , Cs.

Dr . REDL, P. Dr . HOLLÓ, F .

1077 Budapest, Rottenbi l ler utca 50.

Dr . MAGYAR, K. Dr . GYENES, J.

1143 Budapest, Z á s z l ó s utca 31-3a