AN IN VIVO METHOD FOR THE SCREENING OF ANTIFILARIAL AGENTS USING SETARIA CERVI AS TEST ORGANISM K.C. SINGHAL, OM CHANDRA and P.N. SAXENA Department of Pharmacology, J.N. Medical College, A.M.U., Aligarh, India Received for publication September 9, 1971 <I> Setaria cervi</I>(1, 2), a cosmopolitan nematode parasite of cattle, lives in the peritoneal cavity. 85% of cattle are found infected with <I>Setaria cervi</I>.The maximum number of worms (60 to 70 in each head) is found between mid June to August after which the number declines and from December to January incidence is as low as 10 to 20 <I>Setaria cervi</I>per head. From February onwards the number starts increasing. The ratio of male and female in cattles has been found to be 1 : 1.21, but incidence of infection in<I>Setaria these cervi</I> animals is very low. <I>Setaria cervi</I>has been found to be more common in goats and sheep and <I>Setaria cervi</I>in horses. No report is however available regarding the incidence of infection by <I>Setaria cervi</I>in neighbouring countries. Little is known about the life history and mode of transmission, but a blood sucking arthopod vector is presumed to be involved in the completion of the life cycle. Williams (3) succeeded in transplanting these worms from freshly slaughtered cattle to rabbits and showed the presence of microfilariae in the peripheral circulation of the rabbits. Although Williams (3) did not succeed in finding an arthopod vector, his results indicated the possibility of keeping the nematodes alive in an experimental host. Later, Nelson (4) succeeded in transplanting both adult worms and microfilariae to rabbits and monkeys. The morphology of the adult worm and microfilaria is described by Ansari (5). Adult worms measure 10.0±2.5cm (male) and 15.0±2.0cm (female) in length. The microfilariae are microscopic structures 277±14.0 μ long and 7.5±1.5wide. μ Ansari (6) successfully implanted the adult worms intraperi toneally in rats and studied microfilariae periods in the peripheral circulation. Maximum density of the microfilariae in the peripheral blood occurred between 6-8 a.m. and 6-8 p.m. Singhal et al. (7) also successfully transplanted the adult worms of <I>Setaria cervi</I>intraperi toneally in rats and reported disappearance of microfilariae from the peripheral circulation after oral administration of diethylcarbamazine. In the present study an attempt has been made to extend these observations and to establish the value of <I>Setaria cervi</I>as a test organism for the screening of antifilarial agents. MATERIALS AND METHODS Adult Setaria cervi were taken from the peritoneal cavity of freshly slaughtered cattle and put immediately in normal saline which was maintained at the temperature as that of the host. The worms remained alive and actively motile for 8-10 hr after collection. They were implanted 2-3 hr after collection, the time taken to bring them from the slaughter house to the laboratory. Before implantation, worms were given repeated washings with normal saline to free them from extraneous material. Antibiotics were not used in the wash so lution as they affected the worms adversely. Worms were implanted in rats of both sexes, weighing 100-150 g. Each rat was anaesthetised with ether and an incision aboutV" long was made in the abdominal wall. The adult Setar•ia cer°viwere put into the peritoneal cavity. The peritoeum was stitched with No.00 catgut and the body wall with silk thread. An antiseptic was applied to the wound daily to check local infection. The blood of the rats was examined daily for the presence of microfilariae. Thick films were made from 1 ml samples of blood, dried and dehaemoglobinised in distilled water for one hr. Thereafter, the slides were stained with Leishman's stain and microscopically examined for the presence of microfilariae. Rats implanted with four adult worms (2 male+ 2 female) usually showed the presence of microfilariae during the second week after im plantation. Usually 10-20 microfilariae were present in each thick film. In untreated infected animals microfilariae continued to be present for about 6 weeks. The rats remained healthy during this period. To study the periodicity of microfilariae in the peripheral circulation, slides were prepared every 2 hr from 6 A.M. to 8 P.M. Rats which had shown microfilariae in the blood for 3 consecutive days were treated daily with doses of drugs given orally or intraperitoneally (Table 2). Peripheral blood was examined daily for microfilariae and disappearance time was noted. Complete dis appearance of microfilariae from the peripheral circulation for three consecutive days was taken as evidence of antifilarial action. Autopsy was done on half of these rats and adult worms were examined. In the remaining rats, the drug was stopped and the peripheral blood was examined for the reappearance of the microfilariae. If there was no response for fifteen days of medication the drug was considered ineffective and was discontinued. The drugs used were diet hylcarbamazine, piperazine citrate, bephenium hydroxy naphthoate, chloroquine phosphate, tetrachloroethylene and 3-acetamido-4-hydroxyphenyl arsonic acid. RESULTS Two male and two female adult Setar4a cccvi were implanted in rats for the screening of antifilarial agents as this combination was found to cause low mortality and maximum duration of microfilaraemia (54±6 days). When the number of worms implanted was increased, the mortality was higher although microfilariae appeared early in the peripheral blood. When 5 male and 10 female worms were implanted all the rats died within 72 hr (Table 1). Microfilariae appeared in every slide prepared and there was no significant difference in their numbers indicating periodicity at any time between 6 a.m. and 8 p.m. Diethylcarbamazine, the commonly used antifilarial agent, produced complete dis appearance of microfilariae from the peripheral circulation of rats at a dose of 100 mg/kg/day orally, in 4-6 days. The onset time of antifilarial action was delayed 8-10 days and 10 12 days in 50 mg and 25 mg/kg daily doses respectively. The response was 70Y. with 50 mg/kg and 20 % with 25 mg/kg dose. The lower doses were however, ineffective (Table 2). 3-Acetamido-4-hydroxyphenyl arsonic acid was given in doses of 30 and 60 mg/kg/day intraperitoneally. With a 30 mg/kg dose, 40 % of the rats were cleared of microfilariae from peripheral circulation in 2-4 days. However with a higher dose of 50 mg/kg the respones increased to 90 % and time of onset of antifilarial action remained almost the same. Piperazine citrate, tetrachloro-ethylene, bephenium hydroxynaphthoate and chlo TABLE 1. intraperitoneal implantation of adult Setaria cervi in rats and appearance of microfilaria in peripheral circulation. TABLE 2. effect of various chernotherapeutic agents against microfilariae of Setaria cervi in vivo. roquine phosphate were ineffective in their maximum therapeutic doses (See Table 2). As shown in Table 2, 50 , of the rats in which the microfilariae disappeared from the peri pheral circulation after medication were autopsied and out of 140 adult worms obtained from these rats 95 % were found to be actively motile. This indicates that only those compounds which were effective in experimental and human filariasis are able to elimi Setaria cervi from the peripheral circulation by a direct action, and not by killing the adult nate the microfilariae of worms. DISCUSSION Filarial infections are closely specific to their vertibrate hosts, hence, for the screening of antifilarial agents, ideal conditions will be served if human filariasis can be transferred to a closely allied animal host for drug testing. This is not practical with the exception of Brugia malayi in cats (8, 9) and consequently chemotherapeutic screening must be done on a suitably related parasite. In almost all methods presently available it is not completely identical with the human parasite in its reactions to drugs. The best practical procedure to date for the in vivo screening of antifilarial agents involves the use of Litofnosoides carinii infected, cotton rats (10). The validity of this method can be questioned on the basis that L. carinii infection responds to certain drugs which have no effect on human filarial infection produced by Wucherei•ia, Loa loa or Onchocerca. The screening method using Dipetalonerna witei (11) is even less reliable as its chemotherapeutic correspondence to human infection is even less close as compared to L. carinii. Even diethyl carbamazine, a potent filaricidal agent has no effect on D. Witei infection. Furthermore there is a long incubation period of 50 days for Litomosoides carinii (10) and Dipetalonerna witei (11) to 8 months for Dirofilaria iinmitis (12) before microfilariae appear in peripheral circulation. The use of Setaria cervi infection in rats for the screening of antifilarial agents appears to be preferable to the presently available methods. Intraperitoneal implantation of four adult Setaria cervi produced microfilaraemia within 10±3 days and it lasted for 54±6 days which is quite sufficient for screening. If the number of worms implanted is increased the mortality is also increased though the onset of microfilaraemia may be hastened by few days. Four adult worms were therefore implanted throughout the chemotherapeutic trials conducted in the present investigation. The method also fulfils the most important re quirement of a good screening method, in that it corresponds to human filariasis in, its sensitivity to drugs. Therefore, this method which has no arthopod vector as an intermediate host, is easy to carry out, involves little expenditure and time, is a promising method for the screening of antifilarial agents. SUMMARY In the present study, rats were implanted with 4 adult Setaria cervi (2 male and 2 female), a cosmopolitan parasite of cattle, intraperitoneally within 2-3 hr of collection from the slaughter house.