CJap. J. Parasit., Vol. 25, No. 5, 409-414, 1976]

Comparative Studies on the Chemotherapy of Experimental cervi Infection

Abdul BAQUI and Jamil A. ANSARI Department of Zoology, Section of Parasitology, Aligarh Muslim University,

Aligarh (U.P.), India

(Received for publication ; February 16, 1976)

abattoir, were washed several times in Introduction physiological saline to remove extraneous Setaria cervi, the cosmopolitan filariid matter. Implant of the worm into peritoneal worm, has a high rate of incidence among cavity of white rats was made via lapa- buffaloes and cattle population of India. rotomy. Each rat received three female and The parasite is known to produce tissue two male worms. fibrosis, penetration and obstruction in the Blood of each rat was tested for microfi- intestine of the hosts (Sarwar, 1945). Evid lariae, and microfilarial count was done each ences are also available that infective stages day at 6-8 p.m. The amount of blood taken in strange circumstances may enter the was always maintained as 1 mm3. The central nervous system of the natural hosts drugs were administered orally after three and develop to partial maturity causing days of appearance of microfilariae, and severe pathological condition known as continued till the microfilariae disappeared 'lumbar paralysis' (Blazeck et al., 1968; from peripheral blood circulation. Prior to Pachauri, 1972). Accidental infection of this the administration of the drugs, weight of worm is also very common among sheep and each rat was taken. goats wThich may result into ' cerebro-spinal After a week of the discontinuation of nematodiasis \ Suggestions have also been drug, the blood of rats was checked again put forward by Innes (1951), Innes and Shoho to find any possiblity of reappearance of (1952), Kadenatsii (1956), Nelson (1965) that microfilariae. At the close of the experiment, many neurological syndromes of unknown the rats were autopsied. A batch of ten aetiology in man and in tropics are rats non-medicated as control was also kept due to Setaria sp. infection. in the laboratory, and a record on microfila- In view of the heavy incidence of this raemia was maintained. worm and its involvement in serious diseases, the present work of its control by anthel- Results mintics was undertaken. Survival of adult Microfilariae appeared in the peripheral worms for a long duration in alternate hosts blood circulation of all rats with a latent (Williams, 1955 ; Nelson, 1962 ; Ansari, 1964) period of 8±2 days and continued to exist stimulated the screening of anthelmintics in for 65±5 days in control rats. An average rat-cervi system. The drugs used as test maximum density of 19 microfilariae/mm3 chemicals are diethylcarbamazine citrate was recorded in these rats. (Hetrazan), tetramisole and thiabendazole. Use of diethylcarbamazine citrate in the

Materials and Methods experimental rats brought speedy disappea rance of microfilariae from peripheral blood Adult worms collected from the peritoneal circulation. All rats cleared of microfilariae cavity of buffaloes, Bos bubalis from the in 3-6 days at the dose of lOOmg/kg body

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Table 1 Effect of various anthelmintics against microfilariae of Setaria cervi in white rats

Dose Time of Recovery of Duration of Rats mg/kg disappear live adult Drug medication cleared Response body ance of mf. worm on (days) of mf. weight (days) autopsy

Diethyl- 25.0 10-13 12-15 5/10 50% 36% carbamazine 50.0 7-9 9-11 9/10 90% 40% citrate 100.0 3-6 5-8 10/10 100% 48%

Tetramisole 7.5 3-5 5-7 10/10 100% 32%

15.0 2-3 4-5 10/10 100% 28%

Thiabendazole 25.0 Nil 20 0/10 0% 52%

50.0 Nil 20 0/10 0% 48%

100.0 Nil 20 0/10 0% 48%

Untreated — — 0/10 — 40% control —

weight given twice daily presenting a 100 % • CONTROL response. The onset of antifilarial action, kHETRAZAN 0 TETRAMISOLE however, was delayed by 7-9 days and 10- < THIABENDAZOLE /

13 days at the dosages of 50 mg/kg and 25 S 12 mg/kg with 90% and 50% response in respective cases (Table 1). No side effect of any kind was evident at these dosages. Tetramisole, a broad spectrum anthelm- intic, was tested for the first time in the present experiment. The drug showed potent microfilaricidal property. The drug when administered orally in 7.5 mg/kg and 15.0 mg/kg body weight (single dose/day) produced complete elimination of microfila riae from peripheral blood circulation of all rats (response 100%). Disappearance time of microfilariae at 15.0 mg/kg varied from 11 13 15 17 19 21 23 DAYS AFTER INFECTION 2-3 days while slightly delayed by 3-5 days Fig. 1 Efficacy of drugs on the microfilarial at the dose of 7.5 mg/kg. The chemical density in the blood of rats. also showed no side-effect of any kind. The drugs not only brought microfilarial with tetramisole. elimination but the level of microfilaemia Thiabendazole, another broad spectrum also reduced considerably. Maximum micro anthelmintic, when tested in the present filarial density in Hetrazan and tetramisole experiment at various dosages administered treated cases, on an average, varied between twice daily, proved completely ineffective 3.8 and 4.5, and microfilarial population (Table 1). The microfilarial population con showed a declining tendency (Fig. 1). tinued to increase even at higher doses and Microfilariae reappeared after a lapse of one followed the microfilarial curve pattern of week of discontinuation of drug in all control rats (Fig. 1). Hetrazan treated cases whereas no micro The rats were autopsied after the treat filariae were traced in groups of rats treated ment was over. ^Hetrazan treated groups

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of rats were sacrificed over a period of 22- the above observation in part. The drug 30 days from initial infection. Average has shown lethal effect on microfilariae, but recovery of live worms in these rats was remained quite ineffective against the adult 36%, 40% and 48% at the doses of 25 worms in in vivo condition even at higher mg/kg, 50mg/kg and 100 mg/kg respectively. doses. The views expressed by Singhal The remaining worms were either dead, et al. (1973) regarding the decrease in the exhausted or untraceable. Tetramisole treat reproductive potential as indicated by the ed groups were taken up earlier between absence of microfilariae from the blood 22-24 days because of its extreme efficacy. circulation even in the presence of live adult Both of its doses being used produced worms, is based purely on imagination. sufficient mortality, and only 32 % and 28 % Hawking (1950), Hawking et al. (1950) have live worms, on an average, were recovered already shown that microfilariae of Litomo (Table 1). In case of thiabendazole treated soides carinii in the presence of Hetrazan groups, 48-52% of live worms, on an move away, from the blood circulation and average, were recovered from rats even get collected in the liver, and which are after 20 days of continued medication. later destroyed by opsonin-like action. The Average recovery of live worms in untreated characteristic reappearance of microfilariae in control group had been 40% after 30 days the peripheral blood circulation after a week of initial infection. of discontinuation of the drug in the present study is indicative of the fact that the adult Discussion worms have never lost the reproductive potential but continued to produce it through Diethylcarbamazine citrate has been a out, and as the influence of the drug drug of choice in most of the and was over, the microfilariae reappeared in human filariases. Burch and Ashburn (1951), the peripheral blood circulation after a brief Duke (1957, 1968) reported the filaricidal lapse. action of Hetrazan on microfilariae of On- Elaborate studies on the mode of action chocerca volvulus but remained ineffective on adult worms. Cherry (1960) reported He of Hetrazan have been carried out in Litomo trazan as a superior and most effective drug soides, Onchocerca and Wuchereria infections. in onchocerciasis. The allergic reaction, he Hawking (1950) reported that the drug rapidly reported, could be successfully controlled destroys the microfilariae of these worms by by prednisolone. Taylor and Terry (1960) an opsonin-like action. Observations of observed Hetrazan to be ineffective against Taylor (1960) on Litomosoides microfilariae embryos and adult worms of Litomosoides provide perhaps the best possible explanation carinii during in vivo and in vitro studies. on the mode of action of Hetrazan. She However, they reported the drug to be observed that microfilariae under the in effective against microfilariae of Litomosoides fluence of Hetrazan stick to the walls of the carinii. capillaries of the liver by their tails with Singhal et al. (1972, 1973) tested diethyl few leucocytes adhering to their tails. The carbamazine citrate against Setaria cervi in microfilariae, she reported, are probably vivo and in vitro, and found it quite effective eliminated by way of phagocytosis from the against the microfilariae only. They also peripheral blood circulation. Although di observed a decrease in the reproductive ethylcarbamazine appears to be quite effective potential of the adult worms and attributed against microfilariae, but there exists a dose it to be the beneficial effect of the drug. dependent relationship. With the decrease The drug, they reported, was capable of of the dosage, the response also decreases producing 100% mortality of adult worms proportionately. in vitro at a very high dose (750//g/ml The other test chemical, tetramisole, used concentration). The present study supports in this experiment has been in extensive use

( 69 ) 412 as a broad spectrum anthelmintic against a helmintic, when tested on the experimental wide variety of infections (Thien- rats, proved completely ineffective. None of pont et al., 1966; Campbell and Cuckler, 1967 ; the rats cleared of microfilariae even for 20 Bossche and Janssen, 1967; Ross, 1968 ; Thien- days of continued medication. Further, the pont et al., 1969; Moreau and Lagraulet, 1972). recovery of adult worms on autopsy was The drug showed pronounced and mira also 48-52% which are a fairly high figures culous efficacy on the microfilariae of Setaria indicating the inefficacy of the drug in such cervi. The microfilariae, unlike Hetrazan infection. However, Singhal et al. (1973) cases, disappeared completely from peripheral conducted in vitro studies and observed blood circulation at all dosages of tetramisole positive action of thiabendazole against adult administered. Also, the onset of microfila- Setaria cervi and reported 100 % mortality ricidal action was recorded much earlier at a concentration of 300 ^g/ml. The inef than diethylcarbamazine. All rats treated fectiveness of thiabendazole as observed in cleared of microfilariae presenting a 100% the present experiment is undoubtedly in response at all dosages, while in Hetrazan accordance with the earlier report by treated groups the response at various Nnochiri (1966) in patients with Loa loa, dosages was 50%, 90% and 100%. It is Acanthochielonema and Onchocerca infections. also noteworthy that even lower dose of He tested thiabendazole on patients in a tetramisole (7.5 mg/kg) was as much effective dose of 25 mg/kg for 5-10 days and found it as the higher dose (100 mg/kg) of Hetrazan, to be ineffective. Larger doses increased and in both cases response was 100%. the frequency of toxic effects in patients. Although diethylcarbamazine and tetramisole In vivo screening of diethylcarbamazine, are both effective against the microfilariae tetramisole and thiabendazole using Setaria but the latter has definite advantage because cervi as test organism has been very satis of its effectiveness even at lower doses. The factory. Diethylcarbamazine and tetramisole drug has also shown to have some effect on proved to be chemicals of considerable adult worms, and by virtue of which the importance. Both the chemicals have shown percentage of live worms recovered on the capability of destruction of circulating autopsy was only 28-32%, a condition microfilariae in the blood of experimental comparable to untreated control group. host. These could be safely recommended Further, non-reappearance of microfilariae as useful drugs against natural Setaria cervi after a week of discontinuation of drug infection ; and a breakthrough could be reveals the persistence of drug's influence in achieved reducing the risk of transmission. the host body. The drug has been reported Since tetramisole has shown its effectiveness to be tolerable even at several times recom against the adult worms and restricted their mended dose of 2.5 mg/kg (Thienpont et al., life span within the host body, this may be 1969). Ozcan (1967) reported that higher taken as drug of choice. A dual purpose dose of tetramisole such as 20, 30 mg/kg could be served in this way : (1) like Hetrazan body weight produced certain transient it may clear off the microfilariae from blood side-effects in experimental trichinosis in stream preventing its further transmission mice. In vitro study made by Thienpont and (2) may cure the afflicted animals by et al. (1966) reveals that the drug exerts a killing the adult worms. rapid paralysing action on various species of nematode. Moreau and Lagraulet (1972) test Summary ed the in vitro activity of L. tetramisole on the Three important anthelmintics namely third stage larvae of Angiostrogylus canton- diethylcarbamazine citrate, tetramisoJe and ensis and found this chemical very active thiabendazole were tested on white rats even at very low concentration. experimentally infected with Setaria cervi. Thiabendzole, another broad spectrum ant Diethylcarbamazine and tetramisole showed

(70) 413 positive microfilaricidal property. Tetra- on Onchocerca volvulus: I. Methods of as misole has shown detrimental effect on adult sessment, population dynamics of the para worms also which was characterised by the site and the effects of diethylcarbamazine. low percentage of live adult worms recovered Bull. WHO, 39, 137-146. on autopsy. Diethylcarbamazine citrate did 9) Hawking, F. (1950) : Some recent work on not affect adult worms, and also had very filariasis. Trans. Roy. Soc. Trop. Med. Hyg., 44, 153-182. temporary microfilaricidal influence hence 10) Hawking, F., Sewell, P. and Thurston, J. P. there was a reappearance of microfilariae in (1950) : Brit. J. Pharmacol., 5, 217. (Quoted peripheral blood circulation. Thiabendazole by Taylor, A. E. R. (1960) : Trans. Roy. proved completely ineffective on microfilariae Soc. Trop. Med. Hyg., 54, 450-453). as well as adult worms. 11) Innes, J. R. M. (1951) : Necrotising ence- phalomyelitis (or encephalomyelomalacia) of Acknowledgements unknown aetiology in goats in Ceylon with similarities to setariasis of sheep, horses We are highly grateful to Prof. Shah Mashhood and goats in Japan. Brit. Vet. J., 107, 187- Alam, Head, Department of Zoology for provid 203. ing laboratory facilities. Thanks are also due 12) Innes, J. R. M. and Shoho, C.( 1952) : Nema- to the University Grants Commission for financial todes, nervous diseases and neurotropic assistance. virus infection. Observations in animal pathology of probable significance in medical References neurology. Brit. Med. J., 2, 366-368. 13) Kadenatsii, A. N. (1956) : Setariasis in sheep : 1) Ansari, J. A. (1964) : Studies on Setaria biology of causal parasite. C. R. Acad. Sci. cervi (Nematoda : ) Part II. Its USSR, 107, 191-192. peritoneal transplant and periodicity of the 14) Moreau, J. P. and Lagraulet, J. (1972) : Suvie microfilariae in white rats. Z. Parasitenk. in vitro des larves de troisieme stade 24, 105-111. d'Angiostrongylus cantonensis. Etude de 2) Blazeck, K., Dykova, I. and Pav, J. (1968) : l'activite du L. tetramisole dans le milieu The occurrence and pathogenecity of Setaria NCTC 109. Ann. Parasit. Hum. Comp., 47, cervi Rud in the central nervous system 525-529. of a deer. Folia Parasit., Praha, 15, 123- 15) Nelson, G. S. (1962) : Observation on the 130. development of Setaria labiatopapillosa 3) Bossche, V. D. and Janssen, P. (1967) : The using new techniques for infecting biochemical mechanism of action of the ant- aegypti with this nematode. J. Helminth., helmintic drug tetramisole. Life Sci., 6, 36, 281-296. 1781-1782. 16) Nelson, G. S. (1965) : Filarial infections as 4) Burch, T. A. and Ashburn, L. L. (1951) : zoonoses. J. Helminth., 39, 229-250. Experimental therapy of onchocerciasis with 17) Nnochiri, E. (1966) : Thiabendazole in Suramin and Hetrazan : Results of a three human filariasis. Trans. Roy. Soc. Trop. year study. Am. J. Trop. Med., 31, 617-623. Med. Hyg., 60, 601-604. 5) Campbell, W. C and Cuckler, A. C. (1967) : 18) Ozcan, C. (1967) : Koperklerde ascaridosis Comparative studies on the chemotherapy in yeni bir anthelmintik olan tetramisole of experimental trichinosis in mice. Z. ile tedavisi. Vet. Fak. Derg. Ankara Univ., Tropenmed. Parasit., 18, 408-417. 14, 357-362. 6) Cherry, J. K. T. (1960) : The treatment of 19) Pachauri, S. P. (1972) : Cerebrospinal nema- onchocerciasis. East. African Med. J., 37, todiasis in a buffalo. A case report. Indian 550-558. J. Ani. Res., 6, 17-19. 7) Duke, B. O. L. (1957) : The reappearance, 20) Ross, D. B. (1968) : Oral tetramisole. Effect rate of increase and distribution of the on Dictyocaulus viviparus, Ostertagia oster- microfilariae oiOnchocerca volvulus following tagi and Cooperia onchophora in experim treatment with diethylcarbamazine. Trans. entally infected calves. Vet. Record., 83, Roy. Soc. Trop. Med. Hyg., 51, 37-44. 69-71. 8) Duke, B. O. L. (1968) : The effects of drugs 21) Sarwar, M. M. (1945) : On the pathogen-

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ecity of Setaria cervi (Rudolphi, 1819) in The effect of antifilarial drugs on the buffaloes. Curr. Sci., 4, 107. embryonic development of Litomosoides 22) Singhal, K. C, Chandra, O. and Saxena, P. carinii, of the cotton rat. Trans. Roy. Soy. N. (1972) : An in vivo method for the Trop. Med. Hyg., 54, 33-36. screening of antifilarial agents using Setaria 26) Thienpont, D., Vanparijs, O. F. J., Raeyma- cervi as test organism. Japan. J. Pharmacol., ekers, A. H. M., Vandenberk, J., Demoen, 22, 175-179. P. J. A., Allewijn, F. T. N., Marsboom, R. 23) Singhal, K. C, Saxena, P. N. and Johri, M. P. H., Niemegeers, C. J. E. and Janssen, P. B. L. (1973) : Studies on the use of Setaria A. J. (1966) : Tetramisole (R 8299), a new, cervi for in vitro antifilarial screening. potent broad spectrum anthelmintic. Nature, Japan. J. Pharmacol., 23, 793-797. 209, 1084-1086. 24) Taylor, A. E. R. (1960) : Observations with 27) Thienpont, D., Brugmans, J., Abadi, K. and the ultrapak microscope on microfilariae of Tamal, S. (1969) : Tetramisole in the treat Litomosoides carinii circulating in the liver ment of nematode infections in man. Am. of a cotton rat, before and after the admi J. Trop. Med. Hyg., 18, 520-525. nistration of Hetrazan. Trans. Roy. Soc. 28) Williams, H. E. (1955) : Studies on bovine Trop. Med. Hyg., 54, 450-453. Setaria cervi (Rudolphi, 1819). Parasitol., 25) Taylor, A. E. R. and Terry, R. J. (1960) : 45, 56-62.

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