The microfilaricidal effect of ivermectin was studied on Cebus apella infected with Dipetalonema gracile in Paraguay. Nine monkeys were treated with ivermectin at the dosage of 200μg/kg body weight by single subcutaneous injection. The significant reduction in microfilarial count after treatment of ivermectin was recognized (P<0.01). The change of blood picture of 9 monkeys infected with D. gracile following treatment of ivermectin also studied. The total WBC count was found to be significantly increased at 1 day after the treatment. Differential count study showed that neutrophils increased in most monkeys. On the other hand, the significant change in eosinophil and lymphocye count was not observed

Fujita Osamu

Inchausti Alba

Moreno Ricardo A.

Ramona Rosa S.

Rojas Antonieta De Arias

Sakamoto Makoto

Schinini Alicia

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Trop･ Med･, 33 (3), 61者66, September, 1991 61Treatment of Dipetalonema gracile in Cebus apellaby IvermectinMakoto SAKAMOTO2, Osamu FUJITA2, Alicia SCHININI3,Alba INCHAUSTI1, Rosa RAMONA S., Antonieta ROJAS de Arias3and Ricardo MORENOA.3d epartment of Parasitology, Institute of Tropical Medicine, Nagasaki University,12-4, Sakamoto-machi, Nagasaki 852, Japan2Hokkaido Medical Animal Center, N-3, W-24, 259, Chuo-ku, Sapporo, 064, Japan3Instituto de Investigaciones en Ciencias de la Salud, Rio de la Plata y Lagerenza, Asun-cion, 2511, ParaguayAbstract: The microfilaricidal effect of ivermectin was studied on Cebus apella infectedwith Dipetalonema gracile in Paraguay. Nine monkeys were treated with ivermectin at thedosage of 200ƒÊg/kg body weight by single subcutaneous injection. The significant reduc-tion in microfilarial count after treatment of ivermectin was recognized (P<0.01). Thechange of blood picture of 9 monkeys infected with D. gracile following treatment ofivermectin also studied. The total WBC count was found to be significantly increased at 1day after the treatment. Differential count study showed that neutrophils increased in mostmonkeys. On the other hand, the significant change in eosinophil and lymphocye count wasnot observed.Key words: Dipetalonema gracile, Ivermectin, TreatmentINTRODUCTIONIt is well known that Cebus apella, one of the most popular monkey in the New World,harbor Dipetalonematidae in peritoneal cavity (Yagi et al, 1986). However, little is knownabout chemotherapy of this filariasis. In general, diethylcarbamazine (DEC) is widely used inchemotherapy of human and animal filariasis, being particularly effective against microfilaraeof lymphotic filariasis due to Brugia malayi, Brugia timori and Wuchereria bancrofti.However, DEC is not a entire drug for onchocerciasis, subcutaneous filariasis, because of it'ssevere inflammatory reactions though it shows a remarkable microfilaricidal effect. On theother hand, ivermactin, an antihelmintic drug in wide use for several years by the veterinaryindustry, has recently been found to be less side effects and effective against Onchocercavolvulus and W. bancrofti (Greene et al., 1985, Kumaraswani et al., 1988).The present study describes the microfilaricidal effect of ivermectin and inflammatoryreactions elicited by treatment in C. apella infected with Dipetalonema gracile.Received for Publication, September ll, 1991.Contribution No. 2536 from the Institute of Tropical Medicine, Nagasaki University.This study was performed as part of Paraguay/Japan medical cooperation project on Chagas' diseasesupported by Japan International Cooperation Agency (JICA).62MATERIALS AND METHODSTwelve monkeys (3 males and 9 females), C. apella, were provided for this study. All ofthem were captured in the northern part of Paraguay and maintained for more than one yearin the Laboratory of Institute of Health Science, Asuncion University. D. gracile microfilanaewere detected in all of their blood stream･ Microfilarial densities were ranged from 163 to60,784 per O･5 ml of blood. Nine monkeys were treated with ivermectin (Merck, Sharp &Dohme) at the dosage of 200 μg/kg body wieght by single subcutaneous injection･ Threemonkeys were given saline solution through subcutaneous as the control即oup. By theKnott's concentration method, the number of microfilariae per O･5 ml of blood were countedat 1, 2 days and 1, 2, 3, 4 weeks after the initial treatment (Knott, 1930)･To know the inflammatory reaction after treatment, haematological examination wascarried out･ Total number of white blood cell (WBC) per cubic millimeter of blood wascounted by conventional method and differential cell count was assessed under giemsa stain･ing. The blood examination was made between 9:00 and 10:00. Statistical analysis was per-formed where appropriate using Wilcoxon's rank-sum test･RESULTSTable 1 shows the effect of ivermectin on the microfilarial count of D. graale. Meanpercentages of reduction on microfilarial count of trated group at 1, 2, 7, 14, 21 and 28 daysafter the treatment were ･4, 37･4, 49･2, 55･7, 53.3 and 49･7% respectively･ On the otherhand, microfilarial densities of control grooup was not decreased after the injection of salinesolution. The percentage of maximum reduction in microfilarial count of each monkeys rang-Table l･ Microfilarial count in 0.5 ml blood of Cebus apellaNumber of microfilaraeMonkeyNo･Days after treatmentBefore　　　1　　　　2　　　　　7　　　　1 4　　　　21　　　　28P<3oト≡bD'｢ロC)者4者JedA)ゝslsト者)163　　　　　　296, 875　　　1,9991, 068　　　　390503286　　　　　9312, 776　　　6,9882, 179　　　　　6534,432　　　22,41660, 784　　　29, 44012　　　　　　211,331　　1,142443　　　　　5054　　　　　　091　　　　1334,005　　　　5, 30359　　　　　　5023,408　　　21, 64845,216　　　30,96019　　　　　22　　　　　241,572　　　1,328　　　1,991529　　　　　690　　　　　6161　　　　　　2　　　　　　1121　　　　156　　　　1406,480　　　5, 792　　　5, 55259　　　　　52　　　　　6118, 832　　　20, 496　　　20, 77025, 136　　　27,016　　　30, 68810246191273257340245203竜11去…;≡;o4喜･,三97…3,73,3三三4,473,48喜33;…4038喜,4,｡芸…4,03,2冒…63ed from 45.3 to lOO%. One of 9 monkeys showed the maximum reduction in microfilarialcount at 1 day after the treatment. Three monkeys showed the maximum reduction at 2 daysafter the treatment. In other 5 monkeys, the maximum reduction was revealed at 1 and 2weeks after the treatment. A significant decrease in microfilarial count was found at 1, 2, 7,14, 21, 28 days after the treatment of ivermectin (P<0.01). The change of blood picture of 9monkeys infected with D. gratile following treatment of ivermectin was shown in Fig. 1. Thetotal WBC count was found to be increased at 1 day after the treatment･ Seven of 9monkeys showed the significant increase in total number of WBC at 1 day after the treat-ment. The rate of increase varied from 2 to 115 percent of the initial count. On day 2 aftertreatment, the total WBC count returned to the level of initial count. Differential count studyshowed that neutrophils increased in 7 of 9 monkeys with the range from 52 to 425% of theinitial count at 1 day after the treatment. All monkeys with the increase of total WBC show-ed the increase of neutrophil. The significant change in eosinophil and lymphocyte count wasnot observed during this experiment.10′000rl≡≡i?∽いi 5.000いIs0LV.`】=⊃Z!≡≡;iT,¥iた壬.･　　　Total lVBCo　-oNeutrophil■-　Ly川phocyteローロEosinoph月＼iiZS＼IIiii3l､Ⅰi/_ /Io｣ '-sTi-n_∩-ロー｢_∩6　8 10 12 14 1G 18　20　22　2'1 20　28Days after treatmentFig. 1. Changes of blood picture following ivermectin administration.64DISCUSSIONIn this study, ivermectin were firstly tested for it's effectivenss against natural infec-tion of D. gracile in C. apella. Nine monkeys were treated with ivermectin at a single dose of200 jug/kg. The maximumreduction in numbers of microfilariae ranged from 45.3 to 100% ofinitial level. Out of 9 monkeys, 7 showed the maximum,reduction in numbers of microfilariaewithin 1 week after treatment, and these level were maintained over the next 4 weeks. Awad-zi et al. (1985) reported that microfilarial densities fell progressively to reach their lowestlevel at 1 month after the treatment of ivermectin (200 //g/kg) in patient withonchocerciasis, and these level were maintained over next 32 weeks. Soboslay et al. (1987)also reported that abnormal motility was observed in microfilariae in the anterior camber ofpatient with onchocerciasis at 2 days after the treatment of ivermectin. Furthermore, Freed-mannet al. (1989) reported that ivermectin was 100% effective against human ascariasis andstrongyloidiasis on 1 month after treatment.On the other hand, diethylcarbamazice (DEC) was one of the most effective drugsagainst filariasis during recent four decades. Eberhard (1982) described the marked reductionin numbers of microfilariae of D. gracile in squirrel monkeys when treated with DEC (50mg/kg/day for 10 days). However, the numbers of microfilariae had returned to pretreatmentlevel within 4 to 20 weeks. The mechanism(s) of microfilarial killing by DEC were relatedto a variety of condition in host such as complement activation (Hammerberg and Staiunas,1982), activation of granulocytes (King et al., 1983), presence of specific antibody (Sakamotoet al, 1984), and adherence of effector cells to microfilariae (Piessens and Beldekas, 1979;Chandrashekar et al, 1984). On the other hand, Soboslay et al (1987) suggested that ivermec-tin directly impaired motility of microfilariae inducting spastic paralysis. In our study, it wasalso suggested that rapid reduction of microfilariae was caused by direct effect of ivermectin.The inflammatory reaction in patient with filariasis during the treatment of filaricidaldrugs have been discussed. It was generally considered that inflammatory reaction caused byDEC administration was the result of speedy killing of microfilariae. The histological studiesshowed that neutrophils, eosinophils and lymphocytes appeared in close proximity to damag-ed microfilariae (Haqking, 1952, Gibson et al, 1976). These inflammatory reactions may in-duce the change of blood picture of patient. Sakamoto et al (1983) reported that the totalWBC count was found to be rised in patient with onchocerciasis at 3 days after the initialtreatment of DEC. On the other hand, Awadzi et al (1985) reported that there was an in-crease in total WBC counts after day 8, but the levels remained within the normal range.Our study also showed that WBC cont increased (ranged from 2 to 115%) at 1 day after treat-ment, but returned to the initial level at 2 days after treatment. The particular role ofeosinophil also have been discussed in inflammatory reaction caused by filaricidal treatment(Bryceron, 1977; Henson et al, 1979; Guerra-Caceres et al, 1980). Money (1960) reportedthat the number of eosinophil count decreased during the DEC treatment of patient with on-chocerciasis. Guerra-Caceres et al (1980) also described the eosinopenia in patient with on-chocerciasis at 12 hours after the treatment of DEC. When ivermectin was used for patient65with onchocerciasis, Awadzi et al. (1985) reported that there was a slight fall in eosinophilcounts maximal at 36 hours, followed by a steady rise, so that a define increase abovepretreatment levels had occurred by day 8, and by day 28 the level was more than twice theinitial level. In our study, there was no significant change in eosinophil count during the treat-ment of ivermectin in C. apella infected with D. gracile. Although the increase in WBC andneutrophil counts was revealed at 1 day after treatment, the present study suggested thativermectin might not induce severe inflammatory reaction in C. apella infected with D.ACKNOWLEDGMENTWe wish to thank Dra. E. Kasamatsu, chief of Department of Pathology, Institute deInvestigaciones en Ciencias de la Salud, and her colleagues for their kind cooperation. Wealso appreciate Lie. Y. Akimoto for her helpful assistance in this study.REFERENCE S1 ) Awazi, K., Shulz-Key, H., Haddoch, D.R.W., Gilles, H.M. & Aziz, M.A. (1985): The chemotherapyof onchocerciasis X. An assessment of four single treatment regimens of MK-933 (Ivermectin) inhuman onchocerciasis. Ann. Trop. Med. Parasitol, 79, 63-78.2 ) Bryceson, A.D.M., Warrell, D.A. & Pope, H.M. (1977): Dangerous reaction to treatment of onchocer-ciasis with diethylcarbamazine. Brit. Med. J., 1, 742-744.3 ) Chandrashekar, R., Rao, U.R. & Subrahmanyam, D. (1984): Effect of diethylcarbamazine on serumdependent cell-mediated immune reactions to mmicrofilariae in vitro. Tropenmed. Parasit., 35,47-49.4 ) Eberhard, M.L. (1982): Chemotherapy of filariasis in squirrel monkeys. Labo. Ani. Sci., 32,397-400.5 ) Freedman, D.O., Zierdt, W.S., Lujan, A. & Nutman, T.B. (1989): The efficacy of ivermectin in thechemotherapy of gastrointestinal helminthiasis in humans. J. Infect. Dis., 159, 1151-1153.6 ) Gibson, D.W., Conner, D.H., Brown, H.L., Fuglsang, H., Amderson, J., Duke, B.O.L. & Buck, A.A.(1976): Onchocercal dermatitis: Ultrastructural studies of microfilariae and host tissues, before andafter treatment with diethylcarbamazine (Hetrazan). Am. J. Trop. Med. Hyg., 25, 74-87.7 ) Greene, B.M., Taylor, H.R., Cupp, E.W., Murphy, R.T., White, A.T., Aziz, M.A., Shulz-key, H.,D'Anma, S.A., Newland, H.S., Goldschmidt, L.P., Auer, C., Hanson, A.P., Feeman, S.V., Reber,E.W. & William, P.N. (1985): Comparison of ivermectin and diethylcarbamazine in the treatment ofonchocerciasis. N. Engl. J. Med., 313, 133-138.8) Guerra-Caceres, J.G., Bryceson, A.D.M., Quakyi, I. & Spry, C.J.F. (1980): Studies on themechanisms of adverse reactions produced by diethylcarbamazine in patients with onchocer-ciasis-Mazzotti reaction. Parasitol. Immunol, 2, 121- 131.9 ) Hammerberg, B. & Staniunas, RJ. (1982): Diethylcarbamazine-enhanced activation of complementby intact microfilariae of Dirofilaria immitis and their in vitro products. J. Parasit., 65, 809-816.10) Hawking, F. (1952): A histological study of onchocerciasis treated with Hetrazan, Br. Med., 1,992-994.66ll) Henson, P･M･, Mackenzie, C･D･ & Spector, W.G. (1979): Inflammatory reactions in onchocerciasis:A report on current knowledge and recommendation for further study. Bull･ WHO., 57, 667-682･12) King, C.H., Greene, B･M･ & Spagnuolo, P･J･ (1983): Diethylcarbamazine citrate, an antifilarial drug,stimulates human granulocyte adherence林Antimicrob. Agents. Chemother･, 24, 453者456･13) Knott, J. (1930): A method for microfilarial surveys on day blood･ Trans･ Roy･ Soc･ Trop･ Med.Hyg･, 33, 191-197.14) Kumoraswami, V., Ottesen, E林A･, Vijayasekararan, V･, Devi, S･U., Swaminathan, M., Aziz, M.A.,sarma, G.R., Prabhaker, R. & Tripathy, S.P. (1988): Ivermectin for the treatment of Wucherena ban-crofti filariasis. Efficacy and adverse reactions. JAMA., 259, 3150-3153.15) Money, G･L. (1960): `Hetrazan'eosinopenia in onchocerciasis･ A preliminary communication. J･Trop. Med. Hyg., 63, 238-241･16) Piessens, W.F. & Beldekas, N. (1979): Diethylcarbamazine enhances antibody mediated cellularadherence to Bmgia malayi microfilariae. Nature, 282, 845-847.17) Sakamoto, M･ & Zea, G.F･ (1983): The change of blood picture of patients with onchocerciasisfollowing administration of diethylcarbamazine. Trop･ Med･, 25, 47-50.18) Soboslay, P.T., Newland, H.S., White, A.T., Erttmann, K.D., Albiez, E.J., Taylor, H.R., Williams,p･N者& Greene, B.M. (1987): Ivermectin effect on microfilariae of Onchocerca volvulus after a singleoral dose in humans林Trop･ Med･ Parasit･, 38, 8-10林19) Yagi, K., Minezawa, M & Grock, R.T･ (1986): Helminth parasites of Bolivian Cebid monkeys･ Kyotouniversity Overseas Research, Reports of New Monkeys, 6, 51-55･

Treatment of Dipetalonema gracile in Cebus apella by Ivermectin

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Treatment of Dipetalonema gracile in Cebus apella by Ivermectin

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