The effect of metrifonate on Brugia pahangi infections in domestic cats*

Bull. Org mond. Sante 1 1971 45 423-429Bull. Wld Hith Org. ' The Effect of Metrifonate on Brugia pahangi Infections in Domestic Cats* D. A. DENHAM,' T. PONNUDURAI,2 G. S. NELSON,3 FRANCES GUY,4 & ROSEMARY ROGERS5 Metrifonate, an organophosphorus compound, caused a decline in the number of micro- filariae in domestic cats experimentally infected with Brugia pahangi. It was found that the drug was active against both microfilariae and macrofilariae, but probably its greatest effect was on the macrofilarial stages. It is suggested that the drug should be used in filarial infections of man on a trial basis. Studies were carried out to determine the effect of metrifonate, an organophosphorus compound, on Brlugia pahangi in experimentally infected domes- tic cats (Felis catus). This parasite is closely related to the human parasites Brugia malayi and Wuchereria bancrofti, and is widespread in Malaysia in a variety of wild animals. It has been experimentally transmitted to man (Edeson et al., 1960). The life cycle was first studied by Edeson, Wharton & Laing (1960). Third-stage larvae leave the proboscis of the mosquito vector during the act of feeding, penetrate the wound made by the mos- quito, migrate to the lymphatic system of the host, and moult, 8-9 days later, to the fourth stage of the life cycle (Schacher, 1962). The final moult to the fifth stage of the life cycle occurs on the 23rd day in male worms and between days 27 and 33 in female worms (Schacher, 1962). Microfilariae appear in the blood from the 53rd day after infection and 600% of cats show microfilaraemia before day 75; 40 fail to show microfilariae (unpublished data). * Fronm the London School of Hygiene and Tropical Medicine (WHO Reference Centre for Filarial Nematodes). This investigation was supported by the Tropical Medicine Research Board of the United Kingdom, through the Min- istry of Overseas Development, and by Farbenfabriken Bayer AG. 1 Research Fellow and Tutor in Medical Helminthology, London School of Hygiene and Tropical Medicine. 2 Research student, London School of Hygiene and Tropical Medicinie, and lecturer, University of Ceylon. 3Professor of Medical Helminthology, London School of Hygiene and Tropical Medicine. ' Research assistant, London School of Hygiene and Tropical Medicine. 6 Research student, London School of Hygiene and Tropical Medicine. The results of the present experiments suggest that metrifonate is active against the macrofilarial stages of B. pahangi and it is suggested that clinical trials against human filarial infections would be worthwhile. MATERIALS AND METHODS The parasite was obtained from the Liverpool School of Tropical Medicine and is morphologically and biologically indistinguishable from Brugia pahangi as described by Buckley & Edeson (1956). Infective larvae were produced in susceptible Aedes aegypti carrying the fm gene (Macdonald, 1962). The mosquitos were allowed to feed on two consecutive days on a cat with 2-5 microfilariae per mm3 of blood, and 3 days later on a normal guinea pig. Eleven days after the first feed they were immobilized by exposure to a temperature of - 20°C for a few seconds and then killed by percussion. The bodies were placed in a small amount of 199 medium on a large glass slide, and crushed with a test tube. The crushed mosquitos were washed with 199 medium into a Baermann apparatus through a 74-gm pore sieve. After about 2 hours, larvae were removed from the bottom of the apparatus. Cats were infected by injecting known numbers of larvae subcutaneously into the dorsum of the hind foot by means of a 1-ml syringe and a 21-gauge needle. Microfilariae were counted by the chamber counting method described by Denham et al. (1971). The following method was used to recover and count the parasites from the lymphatic system of the cats. After producing deep anaesthesia in the cats with pentobarbital sodium, 0.5 ml of 1% 2737 - 423 - 3 424 D. A. DENHAM AND OTHERS Evans blue in 0.85 %Y saline was injected between the toes of both hind feet. The lymphatic system was stained bright blue by the dye. After 15 minutes, the cat was exsanguinated by opening the chest and cutting one of the major arteries so that during the subsequent dissection bleeding was reduced to a minimum. The limb that had been infected was skinned, the skin lymphatics were cut lengthwise, and the skin was soaked in about 1 litre of warm saline. The popliteal lymph node and its lymph vessels were removed and placed in a dish of 199 medium. The other hind limb was treated in a similar manner. The groin region was skinned and any lymphatic vessels and nodes were removed and placed in a dish of 199 medium. The para- aortic lymph nodes and part of the lymphatic complex of the back were removed and placed in a dish of 199 medium. The hind limbs and the hind region of the body were removed, slashed with a knife, and placed in a jar of warm saline. The lymphatic vessels were carefully dissected and the worms removed. After dissection, the vessels and nodes were carefully examined by com- pression between glass plates to ensure that worms were no longer present and a rough estimate of the number of dead worms present was made. The various pieces removed from the carcass were soaked for 2 hours and then placed together in a bucket of saline and allowed to stand overnight. The fluid in which the pieces had been soaking was poured into a 74-,um pore sieve and the material collected. The worms from the various sources were exposed to 5% metyridine in 199 medium. This treatment caused them to become very relaxed and they could easily be separated from each other, stretched out, and measured. Metrifonate (dimethyl(2,2,2-trichloro-l-hydroxy- ethyl)phosphonate) was obtained from the manu- facturer.' In all experiments, the dosage was 25 mg of metrifonate per kg of body weight and was given either orally by means of a gelatin capsule or by intraperitoneal injection as a 25 mg/ml solu- tion in saline. EXPERIMENTS AND RESULTS Effect ofmetrifonate treatment on microfilarial counts Cats K45 and K46 were each infected with 100 larvae. After 137 days, when it had shown microfilariae in the blood for 83 days, cat K46 was 1 Farbenfabriken Bayer AG, Wuppertal-Elberfeld, Fed- eral Republic of Germany. The ISO common name for this compound is trichlorfon. given 25 mg/kg/day of metrifonate orally on 6 successive days. The results of the microfilarial counts before and after treatment are shown in Fig. 1. Before treatment, counts of around 1 micro- filaria/mm3 were obtained and over the next few weeks they fell to about 0.075 microfilariae/mm3. Thirty-five days after the first series of treatments, the cat was given a single intraperitoneal injection of metrifonate dissolved in saline at a dosage of 25 mg/kg. As can be seen, the counts fell a little more. Cats K68 and K71 were each infected with 100 larvae. Cat K71 was treated with 25 mg/kg/day of metrifonate orally, 113 days after infection and 40 days after becoming patent, on 4 successive days. The results of the counts are shown in Fig. 2. Cat K68 showed a count that increased to around 4 microfilariae/mm3 while the count from cat K71 after treatment was around 0.006 microfilariae/mm3 but still continued at this level for several weeks. The cat was re-treated with 5 doses of the drug orally at 25 mg/kg/day 96 days after the end of the first series of treatments, and for the next 4 months no microfilariae were recovered. Cats K74, K79, and K80 were each infected with 150 larvae; K80 was kept as the untreated control. The other two cats were treated 78 days after infection on 4 days with 25 mg/kg/day orally. Cat K74 had shown microfilariae for 7 days and cat K79 for 14 days at the time of treatment. The counts of microfilariae in all three cats are shown in Fig. 3. The fall in microfilarial counts was not dramatic in either treated cat, but the counts did not rise as they did in the control cat. A second series of 5 daily treatments at 25 mg/kg/day was given 96 days after the first series. Although the counts were reduced to very low levels, micro- filariae were recovered on many occasions over the next 4 months. Effect of metrifonate on developing third-stage larvae in cats Four kittens were each infected with 200 larvae; 2 were kept as untreated controls and the other 2 were given 25 mg/kg/day of metrifonate orally on alternate days. On the day after the second treat- ment, one of the treated kittens died from feline influenza; at autopsy, 2 apparently healthy worms were recovered. The other kitten received 5 doses of the drug. Seven days after the treatment had finished the 3 remaining kittens were killed and the number of living worms that they harboured was EFFECT OF METRIFONATE ON BRUGIA PAHANGI INFECTIONS IN CATS 425 10 /~~~~~~~ -K6 0 ~ ~ %u:~K46 16~~~~~~~~~~~~ % 0.01 100 150 200 250 300 Number of days after infection Fig. 1. Microfilarial counts in cat K45 (untreated control) and cat K46, which was treated with-metrifonate. 10.0~ ~ -- IS .5~~~~~~vN1I /s 20 20 0 Nu-rIfi ay afe riS fcto Fig. 2.Microfiarial ounts i cat K6 (untr ate conrol an cat: K7,w ihwstete ihmtioae D. A. DENHAM AND OTHERS determined. The 2 control cats yielded 33 and 24 worms; no worms were found in the treated animal. Effect of metrifonate on late fourth-stage larvae Four cats were each infected with 200 larvae and 25 days later 2 of them were given a series of 5 daily intraperitoneal injections of metrifonate at a dosage of 25 mg/kg/day; 4 days after the last treatment the four cats were autopsied. Unfortunately, one cat died from feline influenza during the previous night and only dead and partially destroyed worms were seen in the animal. Under similar circumstances we have recovered normal living worms from untreated cats. The two control cats yielded 24 and 29 worms. The treated cat contained 10 living worms and at least 13 dead ones. The mean length of the female worms from the two normal cats was 2.2 and 1.98 cm, respectively, while those from the treated cat were only 1.05 cm long. As only one treated cat was involved, any conclusions from this observation must be very tentative but it is possible that the drug acts as a growth suppressant. Effect of metrifonate on adult worms Four cats were each infected with 200 larvae; 70 days later 2 of them were treated orally with 3 doses of metrifonate at a dosage of 25 mg/kg/day on alternate days. Six days after the last treatment 1 control cat and I treated cat were killed and autopsied. Altogether, 33 adult worms were reco- vered from the control cat while the treated cat yielded 2 normal worms, one of which was alive but very sluggish and covered with a black preci- pitate and some large cell masses, and a large number of dead worms that could not be reliably counted. The other two cats were killed days after treatment; the control cat yielded 16 normal female worms and 8 normal male worms. The treated cat yielded 8 worms, 4 being males, 3 normal females, and I a female with the same type of precipitate and cell adhesions as seen in the other autopsy. Numerous dead worms were seen. Effect of metrifoniate on circulatinsg microfilariae To study this effect, blood was transfused from cats with high levels of microfilariae to normal kittens. It was not possible to transfuse more than 2 kittens on any occasion and the experiment was performed 3 times. In experiment 1, blood was taken from a cat and centrifuged, and the packed red blood cells and microfilariae were injected intra- venously inlto kittens; in experiments 2 and 3, whole blood was transfused. Blood samples Table 1. Number of microfilariae in 100 mm3 of blood of cats given a transfusion of cat blood infected with B. pahangi. In experiment 1 each cat received 100 000 microfilariae per kg, given in packed red cells. In experiments 2 and 3 each cat received 550000 microfilariae per kg in whole blood. Day after trans- fusion 2 3 4 5 6 7 8 9 10 11 12 1 3 14 15 1 6 17 18 19 Experiment 1 Experiment 2 Experiment 3 Normal Treated iNormal ITreated Normal ITreated 42 32 30 8 17 3 9 8 12 36 a 1 o a 6 at 5 a 0 3 3 2 2 0 153 60 102 30 82 103 60 22 18 36 101 a 15 a 38 a 6 a 6 5 -I 8 10 2 3 61 54 12 10 23 43 16 30 12 5 0 50 78 63 10 22 a 38 a 1 0 a o a 2 0 0 a The cat was given 25 mg/kg of metrifonate by intraperitoneal injection on this day. (100 mm3) from the transfused kittens were exa- mined and the number of microfilariae were counted at frequent intervals. Details of the number of microfilariae transfused, the treatment schedule, and counts of microfilariae in the recipient kittens are shown in Table 1. In each case, treatment lowered the number of circulating microfilariae compared with both pre- treatment levels and levels in the control kittens. The surviving microfilariae seemed to live for vir- tually the same time as untreated microfilariae in control kittens. In a study with tetramisole it was found that this drug caused an immediate drop in microfilarial counts followed by a rapid return to normal levels. l l426 EFFECT OF METRIFONATE ON BRUGIA PAHANGI INFECTIONS IN CATS To see if a similar effect occurred in cats treated with metrifonate, microfilarial counts were made before and 1 hour after treatment in two cats. One cat showed counts of 2.8 microfilariae/mm3 before treatment and 0.3 microfilariae/mm3 after treatment: the other showed cotunts of 7.3 and 0.66/mm3. The counts returned to higher levels 1-2 days later. DISCUSSION Several different patterns of microfilarial counts might be seen after treatment with a potentially active antifilarial drug: (1) There might be no change in the microfilarial level; this would indicate that the drug had no useful activity and further investigation in this host-parasite system would be unwarranted. (2) There might be an abrupt drop in the level of microfilariae in the blood, followed by a rise to the previous level. This would suggest that the drug had antimicrofilarial activity but no permanent activity against the adult worms. This drop in the micro- filarial count might be of only short duration, owing to the microfilariae being trapped in the blood capillaries and then released when the drug was either excreted or metabolized to an inactive form by the host, a situation similar to the reversible paralysis of third-stage larvae of Ostertagiai circuni- cincta (Denham, 1970). 10 E C)~~~~~~~I zofl AF %1-. 0.) 15 2 $ E~0- E~~~~~~~~~~~~~ F (3) The microfilarial count might fall slowly and not rise again later. This would indicate that the drug had either killed or sexually sterilized the adult worms. (4) The microfilarial count might fall rapidly and not rise at a later date, indicating that the drug had an effect on both the adults and microfilariae. (5) In filarial infections with marked periodicity, there might be interruption of the normal rhythm; for example, it has been shown that treatment of nocturnally periodic Wuchereria bancrofti with a single dose of diethylcarbamazine has the effect of bringing microfilariae into the peripheral circulation during the daytime (Sasa et al., 1963; Sullivan & Hembree, 1970). Such an effect is unlikely with B. pahangi in cats since this infection shows virtually no periodicity. We have also found that diethyl- carbamazine does not cause a rise in microfilaraemia after treatment of infected cats. Metrifonate caused a substantial reduction in the number of circulating microfilariae of B. pahangi in the cats. The slow decline in the microfilarial counts after treatment seemed to fit the third alternative described above, suggesting that the drug had either sterilized or killed the adult worms. In some cases, however, the effect of the drug was mainly the prevention of the expected rise in microfilarial levels rather than a noticeable reduction in numbers. This Number of days after infection Fig. 3. Microfilarial metrifon ate. counts in cats K80 (untreated control) and cats K74 and K79, which were treated with 427 D. A. DENHAM AND OTHERS was especially true in cats K74 and K79 in which microfilariae had only just started to circulate in detectable numbers (Fig. 3). Although the principal effect of metrifonate in a mature infection seems to be to kill the adult worms, there was evidence of some effect against the micro- filariae. The observations on cats K62 and K67 showed that within an hour of the injection of the drug there was a marked temporary decline in the microfilarial levels; the suggestion being that these drugs paralyse the microfilariae so that they are trapped in the blood capillaries until they recover their normal activity. The experiments in which the cats were transfused with microfilariae and then treated showed that there was a decrease in the number of microfilariae after metrifonate treatment but that treatment did not affect the life span of the surviving microfilariae. The only previous report made of the use of an anthelmintic against B. pahangi or B. malayi in cats is that of Edeson & Laing (1959). They found that diethylcarbamazine killed the adult worms but not the microfilariae. We have confirmed their observa- tion. It is perhaps surprising that diethylcarbamazine should not kill the microfilariae of B. pahangi or B. mnalayi in the cat since the drug is both micro- and macro-filaricidal in W. bancrofti and B. malayi infections in man (Santiago-Stevenson, Oliver- Gonzalez & Hewitt, 1947; Wilson, 1950; Hawking, 1950; Edeson & Wharton, 1958; Ch'en, 1964). The differences between the responses of man and the cat to B. malayi infections when treated with diethylcarbamazine may be due to metabolic varia- tions between the two hosts. Similar differences in response of hosts with the same parasite have been reported by Thompson, Boche & Blair (1968). They found that amodiaquine was active against Litomo- soides carinii in Meriones unguiculatus but was not active against the same strain of the parasite in Siginodoni hispidus. Thomas (personal communica- tion) has also found differences in the effectiveness of diethylcarbamazine, metrifonate, and other com- pounds against Dipetalonema vitei in Praomys natal- ensis and M. persicus. These differences between different hosts may provide further support for the suggestion that metrifonate should be tested in man infected with W. bancrofti and B. malayi, and perhaps the other filarial infections. Metrifonate has been used with considerable success, and with few side effects, in a large number of patients with Schistosoma haematobium infections (see Davis & Bailey (1969) for a recent review). In view of its activity against B. pahangi and its low toxicity (in spite of the lowering of cholinesterase levels) it would be worthwhile testing the drug against W. bancrofti and B. malayi, and other filarial infec- tions in man. As the drug has very little effect on the microfilariae, a more rapid assessment might be made of its effectiveness if the microfilariae were first re- duced by a single treatment with diethylcarbamazine. ACKNOWLEDG&EMENTS We are grateful to Mr Tony Weir for his skilled and interested managerment of the cat colony, and also thank Dr B. W. M. Gooneratne for introducing Briugia pahangi to our laboratory. RESUME ACTION DU METRIFONATE DANS LES INFECr[ONS A BRUGIA PAHfANA,Gl CHEZ LE CHAT DOMESTIQUE Des chats doinestiques infectes expIrimentalement par Brugia pahangi ont ete traites par un organophosphor6, le metrifonate, a la dose quotidienne de 25 mg/kg pendant 4 a 6 jours. Le compose a entraine une reduction imme- diate mais temporaire du nombre de microfilaires suivie, plusieurs semaines plus tard, d'une chute persistante de la mi crofilaremie. L'autopsie a montre la destruction de la quasi-totalite des larves au 3e stade, de nombreuses larves au 4e stade et de la plupart des vers adultes. Des chats ayant requ par transfusion du sang contenant des microfilaires en forte densite ont ete egalement traites par le metrifonate. I1 en est resulte une diminution per- sistante du nombre des microfilaires, mais les larves sur- vivantes ont v&cu aussi longtemps que les microfilaires transfusees a des chats non traites. Ces resultats semblent indiquer que le metrifonate, s'il est actif contre les microfilaires, est avant tout un macrofilaricide. Les auteurs suggerent que le produit fasse l'objet d'essais cliniques pour le traitement des infections filariennes chez l'homme. 428 EFFECT OF METRIFONATE ON BRUGIA PAHANGI INFECTIONS IN CATS 429 REFERENCES Buckley, J. J. C. & Edeson, J. F. B. (1956) J. Helmninth., 30, 1-20 Ch'en, T. T. (1964) Chiin. Med. J., 83, 625-640 Davis, A. & Bailey, B. R. (1969) Bull. Wld Hith Org., 41, 209-224 Denham, D. A. (1970) Exp. Parasit., 28, 493-498 Denham, D. A., Dennis, D. T., Ponnudurai, T., Nelson, G. S. & Guy, F. (1971) Trans. R. Soc. trop. Med. Hyg., 65, 521-526 Edeson, J. F. B. & Laing, A. B. G. (1959) Ann. trop. Med. Parasit., 53, 394-399 Edeson, J. F. B. & Wharton, R. H. (1958) Ann. trop. AMed. Parasit., 52, 87-92 Edeson, J. F. B., Wharton, R. H. & Laing, A. B. G. (1960) Tra,is. R. Soc. trop. Med. Hyg., 54, 439-449 Edeson, J. F. B., Wilson, T., Wharton, R. H. & Laing, A. B. G. (1960) Trans. R. Soc. trop. Med. HiVg., 54, 229-234 Hawking, F. (1950) Trans. R. Soc. trop. Med. Hyg., 44, 153-192 Macdonald, W. W. (1962) Ann. trop. Med. Parasit. 56, 373-382 Santiago-Stevenson, D., Oliver-Gonzalez, J. and Hewitt, R. I. (1947) J. Amer. Med. Ass., 135, 708-712 Sasa, M., Oshima, T., Sato, K., Mitsui, G., Sugato, F., Nishi, S., Yamamoto, H., Tada, I. & Motoi, E. (1963) Jap. J. exp. Med., 33, 213-243 Schacher, J. F. (1962) J. Parasit., 48, 693-706 Sullivan, T. J. & Hembree, S. C. (1970) Trans. R. Soc. trop. Med. Hyg., 64, 787-788 Thompson, P. E., Boche, L. & Blair, L. S. (1968) J. Parasit., 54, 834-837 Wilson, T. (1950) Trats. R. Soc. trop. Med. Hyg., 44, 49-66