EXPERIMENTALPARASITOLOGY 71, 259-266 (1%))
Brugia rnalayi and Brugia pahangi: Transmission Blocking Activity of lvermectin and Brugian Filarial Infections in Aedes aegypfi U. R. RAO,* B. H. KwA,*
J. K. NAYAR,? AND A. C. VICKERY*
*College of Public Health, Department of Environmental and Occupational Health, University of South Florida, Tampa, Florida, U.S.A., and fFlorida Medical Entomology Laboratory, Institute of Food and Agricultural Sciences, University of Florida, Vero Beach, Florida, U.S.A.
RAO, U. R., KWA, B. H., NAYAR, J. K., AND VICKERY, A. C. 1990. Brugia malayi and Brugia pahangi: Transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti. Experimental Parasitology 71, 259-266. Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 pg/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L,, and L, larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L, larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L,‘s recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microtilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microtilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microtilariae to develop to the L, stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microtilariae also failed to develop to L, in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 rig/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microtilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath. Thus, surface alterations by ivermectin might render microfilariae from treated jirds unsuitable for further development. D 1990 Academic PESS, IK. INDEX DESCRIPTORS AND ABBREVIATIONS:Brugia malayi; Brugia pahangi; Aedes aegypti; Ivermectin (Iv); Transmission; Microtilariae (MB; Sausage stage larvae; L, stage larvae; Infective larvae (L,); Infectivity; In vitro feeding; Lectin; Wheat germ agglutinin (WGA); Pokeweed mitogen (PWM); Concanavalin A (Con A); Lentil lectin (LCH); Pisum sativum agglutinin (PSA); Helix pomatia agglutinin (HPA); Soy bean agglutinin (SBA); Dolichos biflorous agglutinin (DBA); Ulex europaeus agglutinin (UEA); Carbohydrate moieties, N-Acetyl glucosamine, D-Mannose; N-Acetyl galactosamine; L-Fucose; Diethylcarbamazine (DEC).
There is increasing evidence that lymphatic filariasis and onchocerciasis are spreading in endemic areas due to the lack of safe, effective chemotherapeutic and prophylactic drugs and to the development of vector resistance to the conventional insecticides. Drugs with efficacy against lymphatic filariasis and onchocerciasis have
been limited to diethylcarbamazine (DEC) and suramin as macrofilaricides. Recently, ivermectin (Iv), which exhibits antinematodal properties (Campbell 1985) has been shown to have greater efficacy against various animal and human filarial parasites (Aziz et al. 1982a, b; Campbell 1987; Kumaraswami et al. 1988) than DEC (Greene et al. 1985; Lariviere et al. 1985). One of the more interesting activities of Iv 259 OOl4-4894190$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
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has been its ability to reduce transmission by interfering with vector competence. In onchocerciasis patients, microfilarial densities are drastically reduced in the skin after a single dose treatment, thus limiting the intake of microfilariae (MI) per fly (Cupp et al. 1986; Bissan et al. 1986; Prod’hon et al. 1987). This long term effect of Iv should significantly control the transmission of the parasite by Simulium vectors. We report here the results of experiments which demonstrate the transmission blocking activity of Iv in A. aegypti fed on jirds parasitized by B. malayi and B. pahangi. The possible mode of action in disrupting the development of Mf to L, and L, in the mosquito is discussed. MATERIALSAND METHODS Animals. Three- to four-week-old male Mongolian jirds (Meriones unguiculatus) were infected with B. pahangi or B. malayi as described by Vickery et al. (1983). Food and water were available ad libitum. Microtilaremic jirds were randomly selected and grouped as control or experimental animals and were used to infect mosquitoes. Ivermectin treatment. Ivermectin (Ivomec, Merck & Co., Inc. Rahway, New Jersey), at the required concentration in sterile distilled water, was prepared fresh before each experiment. Experimental groups of animals (n = 3) were injected subcutaneously with a single dose of the drug at 200 pg/kg body weight. Control jirds (n = 3) were injected with distilled water. Microfilarial counts in 20 pJ of blood from the tail vein were monitored before treatment and prior to the infection of mosquitoes to assess the variations in Mf level after therapy. Mosquito infection. Colonies of Liverpool, Blackeyed A. aegypti were maintained at a temperature of 22-2X with 70-80% relative humidity and fed on cotton pads soaked in 10% sucrose solution. Sugar pads were removed from the cages, 18 to 20 hr before the blood meal. Six- to ten-day-old female mosquitoes, approximately 60 per cage, were infected by allowing them to feed on anaesthetized, infected control (untreated) and experimental (treated) jirds. Mosquitoes were fed on jirds at 2 or 24 hr; 7, 15,30,60, or 90 days after Iv treatment. After the blood meal, mosquitoes were fed on 10% sucrose solution. Mosquitoes were stunned by cooling and individually dissected at different days after the infective blood meal to evaluate the microfilarial intake, sausage stage development, and LZ or L, recovery rates. Experiments were carried
out in duplicate and the results expressed as the number (mean + SE) of developmental or infective stages recovered per mosquito. In vitro bloodfeeding of mosquitoes. Blood was collected from B. malayi-infected microfilaremic jirds by retroorbital bleeding into heparinized tubes. Ivermectin at 50 rig/ml concentration was mixed with the blood and incubated for 30 min at 37°C. Mosquitoes were fed on this treated blood using a membrane feeding technique (Ponnudurai et al. 1971). Control mosquitoes were fed on untreated blood and 14 days later all mosquitoes were dissected individually to recover the L,‘s. Lectin binding to MJ B. malayi and B. pahangi Mf were obtained from peripheral blood of Iv-treated (2 and 24 hr; 7, 15, and 30 days post-treatment) and untreated infected jirds by density gradient centrifugation over isoosmotic Percoll (Sigma Chemical Company, St. Louis, MO) as described by Chandrashekar et al. (1984). The Mf were washed three times in RPM1 1640(GIBCO Diagnostics, Grand Island, NY), pH 7.4, placed into 3-ml polycarbonate tubes, and sedimented by centrifugation at 400g for 10 min using a table top microcentrifuge. Fluoresceinated WGA, PWM, Con A, LCH, PSA, HPA, SBA, DBA, UEA, and their specific inhibitory sugars were obtained from Sigma. Lectin binding assays were carried out essentially as described by Rao et al. (1987a, b). Briefly, 100 pl of FITC-lectins (100 &ml) were added to the pellet containing about 200 Mf and mixed gently. The parasite-lectin mixture was kept in the dark at room temperature (25°C) for 60 min. The Mf suspension was then washed three times with RPM1 1640and a small aliquot was examined under a Leitz fluorescence microscope. The degree of fluorescence was graded as -, negative; +, weak; + +, bright; and + + + , very bright. To ensure the lectin binding specificity, inhibitory sugars (200 mM concentration) were added to the Mf and incubated for 1 hr before the addition of FITC-lectins. Similarly, lectin binding studies were carried out with Mf treated with Iv in vitro. Mf were preincubated with Iv at 50 rig/ml at 37°C for 1 hr and washed with RPM1 1640 before incubating with FITC-lectins.
RESULTS Effect of Iv on circulating Mf of B. malayi and B. pahangi in vivo. Ivermectin,
given as a single dose at 200 kg/kg SC,was ineffective in reducing the circulating microtilaremia of B. pahangi and B. malayi as shown in Fig. 1. Significant suppression of B. malayi but not B. pahangi microfilaremia was observed in treated jirds, only 30 days after treatment. Microfilaremia was
IVERMECTIN
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of mosquitoes fed on jirds 30 days after treatment, when 12 to 14% of mosquitoes harbored B. pahangi larvae. Later, the percentage of mosquitoes infected with B. pahangi increased to 80-87% and was comparable to normal infections. Mosquitoes fed on B. malayi-treated jirds harbored 4-10% L,‘s in comparison to 94100% in the controls. Ivermectin at the tested concentration was not directly toxic to mosquitoes, with less than 10% mortality observed only in groups fed on jirds up to 24 hr after treatment. FIG. 1. Effect of Iv on circulating microtilaremia of B. malayi (iI!, untreated; W, treated) and B. pahangi
(0, untreated; 0, treated) infected jirds.
EfSect of Iv on B. malayi and B. pahangi L, recovery. Mosquitoes fed on Iv-treated B. malayi- and B. pahangi-infected jirds at
different days after treatment were disreduced, 90 days after treatment to an av- sected 14 days post blood meal. The numerage of 65% in B. malayi- and 28% in B. ber of L,‘s recovered per mosquito was calculated and compared with control mosquipahangfinfected jirds. toes as shown in Table I. B. malayi and B. Effect ofZv on infection ofA. aegypti. In pahangi L,‘s were not recovered from mosthis study, the percentage of mosquitoes quitoes fed on treated jirds through 15 days which supported the development of Mf to post-treatment even though these jirds L, after feeding on treated versus untreated were microfilaremic as shown in Fig. 1. jirds was compared (Fig. 2). Initially, all B. pahangi L,‘s were Greater numbers of mosquitoes fed on Iv-treated B. malayiand B. pahangi-infected jirds were free of recovered from mosquitoes fed on microfiL,‘s through 15 days post-treatment. Re- laremic jirds, treated 30 days previously. duction in the percentage of mosquitoes This increase in B. pahangi L,‘s was comwith B. malayi L,‘s was observed in groups parable to controls. In contrast, very few B. malayi L,‘s were recovered from mosquitoes fed on jirds, treated 60 and 90 days previously. 100
..o-- . . . ..= :::.+ ..__ :::::*::::::::;:
. . . . . . . . . . . . . .I’e . . . . . . . . . . . . . ::::::::~~~~8
Effect of Iv on B. malayi L, stage development in A. aegypti. In order to assessthe
efficacy of Iv on the early L, stages, mosquitoes fed on treated B. malayi-infected jirds were dissected on Day 6 after the blood meal. The results (Table II) indicate that Iv completely suppressed the development of Mf to L,‘s in the thoracic muscles of these mosquitoes. Comparison of intake of B. malayi Mf and sausage stage development in mosquitoes. The activity of Iv on the early stages FIG. 2. Infectivity (%) of A. aegypri mosquitoes fed on B. malayi (Cl, untreated; W, treated) and Ii. pahangi (0, untreated; 0, treated) infected jirds.
of larval development immediately after the blood meal was evaluated. As shown in Table III, comparable numbers of Mf were re-
262
RAO ET AL. TABLE I Effect of Iv on L,” Recovery in A. aegypti
Groupb
Hr*/days after treatment
I II
B. malayi
B. pahangi
Experimental
Control
Experimental
Control
2* 2*
0 0
5.88 2 4.24 6.20 k 3.26
8.61 * 1.46 6.42 2 2.13
I II
24* 24*
0 0
4.72 + 3.80 5.24 t 4.14
5.91 + 2.38 4.70 f 2.40
I II
7 I
0 0
3.78 k 3.20 4.10 2 3.80
5.82 k 2.52 6.14 ” 1.29
I II
15 15
0 0
5.16 2 3.80 5.83 f 3.58
0 0
5.28 + 2.32 5.89 2 2.12
I II
30 30
0 0
4.32 f 2.22 5.60 k 3.42
1.68 f 1.53 2.24 2 1.72
6.04 2 0.86 7.10 * 1.20
I II
60 60
0.28 k 0.42 0.32 f 0.50
6.14 f 2.10 5.18 2 1.4
3.2 f 1.60 4.4 2 2.12
5.82 f 3.14 6.27 2 1.56
I II
90 90
0.62 * 0.82 0.54 * 0.86
4.20 2 4.3 6.41 iz 3.6
4.6 2 5.2 5.4 2 3.2
1.28 2 3.24 6.18 ” 2.42
LINumber of L,‘s (mean f SE)/mosquito, 14 days after the blood meal. b Groups I and II represent duplicate experiments. Mosquitoes were fed on one treated or untreated infected jird. Fifty mosquitoes were dissected in each group.
covered from the abdomen of mosquitoes in both experimental and control groups. There was a decrease in the number of Mf recovered from the abdomen 72 hr after the blood meal and no further developmental stages of the parasite were recovered from mosquitoes fed on treated jirds. However, sausage stages were recovered from thoracic muscles of control mosquitoes 72 hr
after feeding on untreated jirds. Microfilariae recovered from the guts of mosquitoes fed on treated jirds were inactive and presumed dead. Effect of Iv treatment on Mf development in mosquitoes fed in vitro. To further evaluate the efficacy of Iv, mosquitoes were allowed to feed on drug-treated microfilaremic jird blood in vitro. Table IV shows that
TABLE II Effect of Iv on B. malayi L,‘s in A. aegypti“
Groupb
Hr*/days after treatment
I II
24* 24*
I II
7 I
Mf/20 (~1(mean f SE) Experimental
Number of L,‘s recovered (mean f SE)
Control
Experimental
Control
88 2 7 92 k 3
72 2 3 81 2 2
0 0
4.92 + 3.56 5.12 + 2.24
104 2 6 94 2 8
96 2 5 9024
0 0
8.14 + 2.86 7.86 * 4.20
0 Mosquitoes (n = 50) were dissected on the 6th day post blood meal. b Groups I and II represent duplicate experiments. Mosquitoes were fed on one treated or untreated infected jird. Mf, Microtilariae from drug treated (experimental) and untreated (control) jird blood.
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PARASITES
TABLE III Comparison of Intake of Microfilariae (MB and Sausage Stage (S) Development in A. aegypti” Fed on Iv-Treated B. malayi Infected Jird Hr after Infection
Experimental
Control
Group”
Head
Thorax
Abdomen
Head
Abdomen
24
I II
0 0
0 0
119 2 29(MD 88 2 16 (M~J
0 0
0 0
64kll 58 -t 8
48
I II
0 0
0 0
72 -t 10 (M~J 23 t 11 (M~J
0 0
0 0
36 + 12 (Mf) 42 + 8 (MD
72
I II
0 0
0 0
17 + 10 (Mf) 0
0 0
6 2 3 (S) 9 + 2 (S)
0.6 2 0.8 (Mt) 0
%
I II
0 0
0 0
0 0
0 0
7 + 3 (S) 10 + 2 (S)
2 k 6 (M~J 0.4 k 0.2 (Mt)
120
I II
0 0
0 0
0 0
0 0
8 + 4 (S) 6 + 5 (S)
0 0
ThOEWi
(Mf) (Mf)
y Fifty mosquitoes were dissected in each group. Mosquitoes were fed on one treated or untreated infected jird. b Groups I and II represent duplicate experiments.
L,‘s of B. malayi were not recovered from the experimental group even though comparable numbers of Mf were recovered from control and experimental groups of mosquitoes, 24 hr after the blood meal. Effect of Iv on lectin binding to Mf. The results in Table V show patterns of lectin binding to B. malayi and B. pahangi Mf recovered from Iv-treated and untreated jirds. These data indicate that Iv treatment causes surface membrane changes with new carbohydrate components such as Nacetyl galactosamine, D-mannose, and Lfucose, exposed on the surface in addition
to N-acetyl glucosamine. Very bright fluorescence was observed with FITC-WGA and -PWM, compared to Con A, HPA, and PSA. Lentil lectin, SBA, DBA, and UEA bound weakly to the sheath surface of Mf. The binding was completely inhibited by the competing sugars at 200 mM concentration. This pattern of altered carbohydrate complexities was observed on Mf recovered from jirds through 15 days after Iv treatment. Microfilariae recovered 30 days after treatment showed only N-acetyl glucosamine, similar to normal Mf from untreated jirds. Microfilariae preincubated
TABLE IV Effect of Iv” in in Vitro Feeding Experiments Using B. malayi Microtilariae
Number of mosquitoes dissected
Number of Mf recovered 24 hr post blood meal/mosquito
Number of L, recovered 14 days after blood meal/mosquito
Mosquitoes infected (%o) with L,
Experimental (treated): 110*2 I II 70 2 4
25 25
48 2 12 24 t- 8
0 0
-
Control (untreated): I 114 % 6 II 84 + 3
25 25
36 t IO 28 2 7
6.2 2 3.1 5.3 + 4.2
Groupb
Mf/20 ~1 of blood
68 76
a Iv (50 &ml) was added to jird blood containing B. malayi Mf for in vitro membrane feeding of mosquitoes. b Groups I and II are duplicate experiments. Results are represented as means 2 SE of larval stages.
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RAO ET AL. TABLE V Effect of Iv on Lectin Binding Characteristics of B. malayi and B. pahangi Microfilariae Mf from jirds’
LectinsO WGA
Specificity
Con-A
N-Acetyl glucosamine N-Acetyl glucosamine o-Mannose
LCH
D-Mannose
PSA
D-Mannose
HPA
N-Acetyl galactosamine N-Acetyl galactosamine N-Acetyl galactosamine L-Fucose
PWM
SBA DBA UEA
Inhibitory sugarsb + + + + + + + + +
Treated
Untreated
Bm
BP
Bm
BP
+++ +++ ++ + ++ ++ + + + -
+++ +++ ++ + ++ ++ + + + -
+++ +++ -
+++ +++ -
-
-
-
-
-
-
-
-
-
-
-
-
a FITC-lectins were used at 100 &ml concentration. b Corresponding inhibitory sugars were added at 200 mM concentration. ’ Mf (Bm, B. mafuyi; Bp, B. pahangi) from jirds were isolated and tested for lectin binding activity 7 days after Iv treatment). Degree of Fluorescence: - , negative; + , weak; + + , bright; + + + , very bright.
with Iv in vitro, however, failed to show the additional carbohydrate moieties (data not shown).
1988). Ivermectin also appeared to reduce Mf counts of subperiodic B. malayi in leaf monkeys (Presbytis cristata) (Mak et al. 1988). Similarly, marked Mf reduction was DISCUSSION observed in Mastomys natalensis infected Ivermectin, a semisynthetic macrocyclic with B. malayi (G. Lammler and I. Sanger, lactone, has been shown to have activity as unpublished observations, quoted by Camp an antitilarial agent (Campbell 1985, 1987). bell, 1982). Ivermectin was, however, Several field studies involving Iv treatment found to be ineffective in reducing the Mf of of human subjects infected with On- B. pahangi in M. unguiculatus or Felis cachocerca volvulus suggested that Iv re- tus (Denham 1982). Studies with 0. volvuduces skin microfilaremia and thus inter- lus, Litomosoides carinii, and Acanrupts the transmission of the parasite to its thocheilonema viteae suggested that Iv ininsect vector (Cupp et al. 1986, 1989; terfered with embryogenesis and the Prod’hon ef al. 1987). Efficacy of Iv on Mf release of Mf from adult worms (Awadzi et of lymphatic filarial parasites is varied de- al. 1985; Schulz-Key et al. 1986; Zahner et pending upon the species. Previously, Iv al. 1987). In this study, Iv appeared to have was found to be completely effective in a long term effect on the subsequent develclearing the blood Mf of Wuchereria ban- opment of Mf in the mosquito vector. Becrofti within 5 to 12 days with reappearance cause the half life of Iv is between 2 and 8 of Mf after 3 months (Kumaraswami et al. days (Bennett et al. 1988; Chiu et al. 1986)
IVERMECTIN
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and is absorbed effectively into the adult worm tissues (R. E. Howells, unpublished, quoted by Campbell 1987) the drug would have sufficient time to act on an entire population of Mf. It is evident from the results of our study that Iv does not suppress the microtilaremia of B. malayi and B. pahangi in jirds for a period after treatment. Brugia malayi circulating Mf started decreasing only 30 days after treatment and B. pahangi Mf levels were unchanged. Interestingly, A. aegypti mosquitoes fed on microfilaremic jirds, 1530 days post-treatment never became infective. The ingested Mf from these treated animals failed to develop either to the sausage stage, L,‘s, or L,‘s. Thirty days after a single dose treatment B. pahangi mosquito infectivity was reestablished. Sixty and ninety days post-treatment, the number of B. pahangi L,‘s recovered from the experimental group of mosquitoes and the percentage of infected mosquitoes were comparable to controls. In contrast, mosquitoes fed on B. malayi jirds showed comparatively fewer L,‘s even at 60 and 90 days post-treatment and only a small percentage of mosquitoes were infected. The results of in vitro feeding experiments further corroborated these in vivo findings by demonstrating the failure of Mf to develop to L,‘s. These results support previously reported observations that B. pahangi infections in cats or Meriones are not susceptible to Iv (Denham 1982) compared to B. malayi infections in M. natalensis (Campbell 1982). Correspondingly lesser effects on the infectivity of mosquitoes fed on Iv-treated B. pahangi jirds were observed in the present study. In addition to the ability of Iv to reduce circulating microtilaremia, as reported earlier, an alternative mechanism of Iv activity is suggested by the results of the present study, i.e., biochemical alteration of the Mf surface. Such changes have been previously suggested to affect Mf development in the insect vector (Furman et al. 1983;
265
Devaney 1985). We found changes in the surface lectin binding properties of Mf recovered from jirds through 15 days posttreatment. Additional sugar moieties like Dmannose, N-acetyl galactosamine, and Lfucose were identified on the sheath surface of Mf exposed to Iv in vivo. In contrast, the present and previously reported studies with in vitro incubations of Mf with Iv and or DEC (Nwachukwu et al. 1987; Devaney 1985; Rao et al. 1987a, b) failed to demonstrate changes in lectin binding properties of Mf. It may be possible that our observations regarding the in vivo activity of Iv are the results of synergistic activities of host components with the drug. Further studies to elucidate in vivo drug interactions are being conducted to extend our knowledge about the transmission blocking activity of Iv. ACKNOWLEDGMENTS We thank Dr. Charles H. Courtney, Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville, Florida for providing us a sample of Ivomec and Mrs. Robin Buck for excellent technical assistance. U.R.R. was supported by funds from the Filariasis component of the UNDP/ World Bank/WHO special program for Research and Training in Tropical Diseases. The study was supported in part by research Grant AI 20052 awarded by the NIAID of the NIH. REFERENCES AWADZI, K., DADZIE, K. Y., SCHULZ-KEY, H., HADDOCK, R. W., GILLES, H. M., AND AZIZ, M. A.
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O., DEMBELE, D., GINOUX, J., COZETTES, P., AND RDANQUE, P. 1986. L’ivermectine (MK-933) dans le traitement de l’onchocercose, son incidence sur la transmission d’Onchocerca volvulus en savane soudanienne an Mali. Medecine d’ Afrique Noire 33, 81-93. CAMPBELL, W. C. 1982. Efficacy of the avermectins against filarial parasites: A short review. Veterinary Research Communications 5, 251-262. CAMPBELL, W. C. 1985. Ivermectin: An update. Parasitology Today 1, 10-16. CAMPBELL, W. C. 1987. Ivermectin versus Nematodes. Molecular Paradigms for Eradicating Helminthic Parasites, pp. 515-524. A. R. Liss, Inc. CHANDRASHEKAR, R., RAO, U. R., RAIASEKARIAH, G. R., AND SUBRAHMANYAM, D. 1984. Separation of viable microfilariae free of blood cells on Percoll 58, 69-70. gradients. Journal of Helminthology CHIU, L. S., SESTOKAS, E., TAUB, R., BUHS, R. P., GREEN, M., SESTOKAS, R., VANDENHEUVEL, W. J. A., ARISON, B. H., AND JACOB, T. A. 1986. Metabolic disposition of ivermectin in tissues of cattle, sheep, and rats. Drug Metabolism And Disposition 14, 590-600. CUPP, E. W., BERNARDO, M. J., KISZEWSKI, A. E., COLLINS, R. C., TAYLOR, H. R., AZIZ, M. A., AND GREENE, B. M. 1986. The effects of ivermectin on transmission of Onchocerca volvulus. Science 231, 740-742. CUPP, E. W., ONOFRE OCHOA, A., COLLINS, R. C., RAMBERG, F. R., AND GUILLERMO ZEA, F. 1989. The effect of multiple ivermectin treatments on infection of Simulium ochraceum with Onchocerca volvulus. American Journal of Tropical Medicine and Hygiene 40, 501-506. DENHAM, D. A. 1982. The effects of some avermectins on the growth of Brugia pahangi. Methods and Findings in Experimental and Clinical Pharmacology 4, 347450. DEVANEY, E. 1985. Lectin-binding characteristics of Brugia pahangi microfilariae. Tropical Medicine and Parasitology 36, 25-28. FURMAN, A., AND ASH, L. R. 1983. Analysis of Brugia pahangi microfilariae surface carbohydrates: Comparison of the binding of a panel of fluoresceinated lectins to mature in vivo-derived and immature in-utero derived microtilariae. Acta Tropica 40, 45-5 1. GREENE, B. M., TAYLOR, H. R., CUPP, E. W., MURPHY, R. P., WHITE, A. T., AZIZ, M. A., SCHULZKEY, H., D’ANNA, S. A., NEWLAND, H. S., GOLDSCHMIDT, L. P., AUER, C., HANSON, A. P., FREEMAN, S. V., REBER, AND WILLIAMS, P. N. 1985. Comparison of ivermectin and diethylcarbamazine in the treatment of onchocerciasis. New England Journal of Medicine 313, 133-138. KUMARASWAMI, V., OTTESEN, E. A., VIJAYASEKARAN, V., UMA DEVI, S., SWAMINATHAN,
M., Azrz, M. A., SARMA, G. R., PRABHAKAR, R., AND TIUPATHY, S. P. 1988. Ivermectin for the treatment of Wuchereria bancrofti filariasis. Efficacy and adverse reactions. The Journal of The American Medical Association 259, 3150-3153. LARIVIERE, M., VINGTAIN, P., AZIZ, M., BEAUVAIS, B., WEIMANN, D., DEROUIN, F., GINOUX, J., SCHULZ-KEY, H., GAXOTTE, P., BASSET, D., AND SARFATI, C. 1985. Double blind study of ivermectin and diethylcarbamazine in African onchocerciasis patients with ocular involvement. Lancet 2, 174177. MAK, J. W., LAM, P. L. W., NOOR RAIN, A., AND SURESH, K. 1988. Effect of ivermectin against subperiodic Brugia malayi infection in the leaf monkey, Presbytis cristata. Parasitology Research 14, 383385. NWACHUKWU, M. A., MACKENZIE, C. D., LITCHFIELD, T. M., AND HOWARD, G. 1987. Lectin binding to the developing forms of Onchocerca gutturosa microfilariae. Tropical Medicine and Parasitology 38, 131-134. PONNUDURAI, T., DENHAM, D. A., AND NELSON, G. S. 1971. The use of a membrane feeding technique for infecting mosquitoes with tilarial worms transported between labortories. Journal of Helminthology XLV, 415418. PROD’HON, J., LARDEUX, F., BAIN, O., HEBRARD, G., AND PRU’DHOM, J. M. 1987. Ivermectine et modalities de la reduction de l’infection des simulies dans un foyer forestier d’onchocercose humaine. Annales de Parasitologie Humaine et Comparee 62, 590598. RAO, U. R., CHANDRASHEKAR, R., PARAB, P. B., RAJASEKARIAH, G. R., AND SUBRAHMANYAM, D. 1987a. Lectin binding characteristics of Wuchereria bancrofti microfilariae. Acta Tropica 44, 35-42. RAO, U. R., CHANDRASHEKAR, R., AND SUBRAHMANYAM, D. 1987b. Litomosoides carinii: Characterization of surface carbohydrates of microtilariae and infective larvae. Tropical Medicine and Parasitology 38, 15-18. SCHULZ-KEY, H., GREENE, B. M., AWADZI, K., LARIVIERE, M., KLAGER, S., DADZIE, Y., AND AZIZ, M. A. 1986. Efftcacy of ivermectin on the reproductivity of female Onchocerca volvulus. Tropical Medicine and Parasitology 37, 89. VICKERY, A. C., VINCENT, A. L., AND SODEMAN, JR. W. A., 1983. Effect of immune reconstitution on resistance to Brugia pahangi in congenitally athymic nude mice. Journal of Parasitology 69,47w85. ZAHNER, H., SKNGER, I., LKMMLER, G., AND MULLER. H. A. 1987. Effect of ivennectin in Dioetalonema viteae and Litomosoides carinii infections of Mastomys natalensis. Tropical Medicine and Parasitology 38, 117-122. Received 26 September 12 March 1990
1989; accepted with revision
Brugia rnalayi and Brugia pahangi: Transmission Blocking Activity of lvermectin and Brugian Filarial Infections in Aedes aegypfi U. R. RAO,* B. H. KwA,*
J. K. NAYAR,? AND A. C. VICKERY*
*College of Public Health, Department of Environmental and Occupational Health, University of South Florida, Tampa, Florida, U.S.A., and fFlorida Medical Entomology Laboratory, Institute of Food and Agricultural Sciences, University of Florida, Vero Beach, Florida, U.S.A.
RAO, U. R., KWA, B. H., NAYAR, J. K., AND VICKERY, A. C. 1990. Brugia malayi and Brugia pahangi: Transmission blocking activity of ivermectin and brugian filarial infections in Aedes aegypti. Experimental Parasitology 71, 259-266. Brugia malayi- or Brugia pahangi-infected, microfilaremic jirds (Meriones unguiculatus) were treated with ivermectin at a single dose of 200 pg/kg body weight, administered subcutaneously. After different time intervals, Aedes aegypti mosquitoes were fed on treated or untreated jirds. Sausage stage, L,, and L, larvae failed to develop in mosquitoes that fed on jirds from 15 to 30 days post-treatment. After 1 month, the numbers of L, larvae recovered from mosquitoes fed on treated B. pahangi jirds were comparable to controls. However, the number of L,‘s recovered from mosquitoes fed on B. malayi jirds remained significantly lower than controls, 2 and 3 months after treatment. This reduction suggests that ivermectin may be more effective in blocking transmission of B. malayi than B. pahangi. Ivermectin treatment had no effect on the mean number of circulating microtilariae in treated jirds. Therefore, mosquitoes ingested comparable numbers of microtilariae when compared to those mosquitoes fed on untreated controls. Only in the case of jirds infected with B. malayi did the circulating microfilarial counts fall 30 days after treatment. The failure of microtilariae to develop to the L, stage in mosquitoes fed on jirds within 30 days of treatment was not due to failure of mosquitoes to ingest microfilariae. Brugia malayi microtilariae also failed to develop to L, in mosquitoes that were allowed to feed on microfilaremic jird blood treated with ivermectin (50 rig/ml) in vitro, indicating its efficacy at low concentrations. In addition to N-acetyl glucosamine, microtilariae obtained for a period of 15 days from ivermectin-treated but not control jirds showed D-mannose, N-acetyl galactosamine, and L-fucose moieties on the surface of the sheath. Thus, surface alterations by ivermectin might render microfilariae from treated jirds unsuitable for further development. D 1990 Academic PESS, IK. INDEX DESCRIPTORS AND ABBREVIATIONS:Brugia malayi; Brugia pahangi; Aedes aegypti; Ivermectin (Iv); Transmission; Microtilariae (MB; Sausage stage larvae; L, stage larvae; Infective larvae (L,); Infectivity; In vitro feeding; Lectin; Wheat germ agglutinin (WGA); Pokeweed mitogen (PWM); Concanavalin A (Con A); Lentil lectin (LCH); Pisum sativum agglutinin (PSA); Helix pomatia agglutinin (HPA); Soy bean agglutinin (SBA); Dolichos biflorous agglutinin (DBA); Ulex europaeus agglutinin (UEA); Carbohydrate moieties, N-Acetyl glucosamine, D-Mannose; N-Acetyl galactosamine; L-Fucose; Diethylcarbamazine (DEC).
There is increasing evidence that lymphatic filariasis and onchocerciasis are spreading in endemic areas due to the lack of safe, effective chemotherapeutic and prophylactic drugs and to the development of vector resistance to the conventional insecticides. Drugs with efficacy against lymphatic filariasis and onchocerciasis have
been limited to diethylcarbamazine (DEC) and suramin as macrofilaricides. Recently, ivermectin (Iv), which exhibits antinematodal properties (Campbell 1985) has been shown to have greater efficacy against various animal and human filarial parasites (Aziz et al. 1982a, b; Campbell 1987; Kumaraswami et al. 1988) than DEC (Greene et al. 1985; Lariviere et al. 1985). One of the more interesting activities of Iv 259 OOl4-4894190$3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
260
RAO ET AL.
has been its ability to reduce transmission by interfering with vector competence. In onchocerciasis patients, microfilarial densities are drastically reduced in the skin after a single dose treatment, thus limiting the intake of microfilariae (MI) per fly (Cupp et al. 1986; Bissan et al. 1986; Prod’hon et al. 1987). This long term effect of Iv should significantly control the transmission of the parasite by Simulium vectors. We report here the results of experiments which demonstrate the transmission blocking activity of Iv in A. aegypti fed on jirds parasitized by B. malayi and B. pahangi. The possible mode of action in disrupting the development of Mf to L, and L, in the mosquito is discussed. MATERIALSAND METHODS Animals. Three- to four-week-old male Mongolian jirds (Meriones unguiculatus) were infected with B. pahangi or B. malayi as described by Vickery et al. (1983). Food and water were available ad libitum. Microtilaremic jirds were randomly selected and grouped as control or experimental animals and were used to infect mosquitoes. Ivermectin treatment. Ivermectin (Ivomec, Merck & Co., Inc. Rahway, New Jersey), at the required concentration in sterile distilled water, was prepared fresh before each experiment. Experimental groups of animals (n = 3) were injected subcutaneously with a single dose of the drug at 200 pg/kg body weight. Control jirds (n = 3) were injected with distilled water. Microfilarial counts in 20 pJ of blood from the tail vein were monitored before treatment and prior to the infection of mosquitoes to assess the variations in Mf level after therapy. Mosquito infection. Colonies of Liverpool, Blackeyed A. aegypti were maintained at a temperature of 22-2X with 70-80% relative humidity and fed on cotton pads soaked in 10% sucrose solution. Sugar pads were removed from the cages, 18 to 20 hr before the blood meal. Six- to ten-day-old female mosquitoes, approximately 60 per cage, were infected by allowing them to feed on anaesthetized, infected control (untreated) and experimental (treated) jirds. Mosquitoes were fed on jirds at 2 or 24 hr; 7, 15,30,60, or 90 days after Iv treatment. After the blood meal, mosquitoes were fed on 10% sucrose solution. Mosquitoes were stunned by cooling and individually dissected at different days after the infective blood meal to evaluate the microfilarial intake, sausage stage development, and LZ or L, recovery rates. Experiments were carried
out in duplicate and the results expressed as the number (mean + SE) of developmental or infective stages recovered per mosquito. In vitro bloodfeeding of mosquitoes. Blood was collected from B. malayi-infected microfilaremic jirds by retroorbital bleeding into heparinized tubes. Ivermectin at 50 rig/ml concentration was mixed with the blood and incubated for 30 min at 37°C. Mosquitoes were fed on this treated blood using a membrane feeding technique (Ponnudurai et al. 1971). Control mosquitoes were fed on untreated blood and 14 days later all mosquitoes were dissected individually to recover the L,‘s. Lectin binding to MJ B. malayi and B. pahangi Mf were obtained from peripheral blood of Iv-treated (2 and 24 hr; 7, 15, and 30 days post-treatment) and untreated infected jirds by density gradient centrifugation over isoosmotic Percoll (Sigma Chemical Company, St. Louis, MO) as described by Chandrashekar et al. (1984). The Mf were washed three times in RPM1 1640(GIBCO Diagnostics, Grand Island, NY), pH 7.4, placed into 3-ml polycarbonate tubes, and sedimented by centrifugation at 400g for 10 min using a table top microcentrifuge. Fluoresceinated WGA, PWM, Con A, LCH, PSA, HPA, SBA, DBA, UEA, and their specific inhibitory sugars were obtained from Sigma. Lectin binding assays were carried out essentially as described by Rao et al. (1987a, b). Briefly, 100 pl of FITC-lectins (100 &ml) were added to the pellet containing about 200 Mf and mixed gently. The parasite-lectin mixture was kept in the dark at room temperature (25°C) for 60 min. The Mf suspension was then washed three times with RPM1 1640and a small aliquot was examined under a Leitz fluorescence microscope. The degree of fluorescence was graded as -, negative; +, weak; + +, bright; and + + + , very bright. To ensure the lectin binding specificity, inhibitory sugars (200 mM concentration) were added to the Mf and incubated for 1 hr before the addition of FITC-lectins. Similarly, lectin binding studies were carried out with Mf treated with Iv in vitro. Mf were preincubated with Iv at 50 rig/ml at 37°C for 1 hr and washed with RPM1 1640 before incubating with FITC-lectins.
RESULTS Effect of Iv on circulating Mf of B. malayi and B. pahangi in vivo. Ivermectin,
given as a single dose at 200 kg/kg SC,was ineffective in reducing the circulating microtilaremia of B. pahangi and B. malayi as shown in Fig. 1. Significant suppression of B. malayi but not B. pahangi microfilaremia was observed in treated jirds, only 30 days after treatment. Microfilaremia was
IVERMECTIN
AND
TRANSMISSION
OF BRUGIAN
PARASITES
261
of mosquitoes fed on jirds 30 days after treatment, when 12 to 14% of mosquitoes harbored B. pahangi larvae. Later, the percentage of mosquitoes infected with B. pahangi increased to 80-87% and was comparable to normal infections. Mosquitoes fed on B. malayi-treated jirds harbored 4-10% L,‘s in comparison to 94100% in the controls. Ivermectin at the tested concentration was not directly toxic to mosquitoes, with less than 10% mortality observed only in groups fed on jirds up to 24 hr after treatment. FIG. 1. Effect of Iv on circulating microtilaremia of B. malayi (iI!, untreated; W, treated) and B. pahangi
(0, untreated; 0, treated) infected jirds.
EfSect of Iv on B. malayi and B. pahangi L, recovery. Mosquitoes fed on Iv-treated B. malayi- and B. pahangi-infected jirds at
different days after treatment were disreduced, 90 days after treatment to an av- sected 14 days post blood meal. The numerage of 65% in B. malayi- and 28% in B. ber of L,‘s recovered per mosquito was calculated and compared with control mosquipahangfinfected jirds. toes as shown in Table I. B. malayi and B. Effect ofZv on infection ofA. aegypti. In pahangi L,‘s were not recovered from mosthis study, the percentage of mosquitoes quitoes fed on treated jirds through 15 days which supported the development of Mf to post-treatment even though these jirds L, after feeding on treated versus untreated were microfilaremic as shown in Fig. 1. jirds was compared (Fig. 2). Initially, all B. pahangi L,‘s were Greater numbers of mosquitoes fed on Iv-treated B. malayiand B. pahangi-infected jirds were free of recovered from mosquitoes fed on microfiL,‘s through 15 days post-treatment. Re- laremic jirds, treated 30 days previously. duction in the percentage of mosquitoes This increase in B. pahangi L,‘s was comwith B. malayi L,‘s was observed in groups parable to controls. In contrast, very few B. malayi L,‘s were recovered from mosquitoes fed on jirds, treated 60 and 90 days previously. 100
..o-- . . . ..= :::.+ ..__ :::::*::::::::;:
. . . . . . . . . . . . . .I’e . . . . . . . . . . . . . ::::::::~~~~8
Effect of Iv on B. malayi L, stage development in A. aegypti. In order to assessthe
efficacy of Iv on the early L, stages, mosquitoes fed on treated B. malayi-infected jirds were dissected on Day 6 after the blood meal. The results (Table II) indicate that Iv completely suppressed the development of Mf to L,‘s in the thoracic muscles of these mosquitoes. Comparison of intake of B. malayi Mf and sausage stage development in mosquitoes. The activity of Iv on the early stages FIG. 2. Infectivity (%) of A. aegypri mosquitoes fed on B. malayi (Cl, untreated; W, treated) and Ii. pahangi (0, untreated; 0, treated) infected jirds.
of larval development immediately after the blood meal was evaluated. As shown in Table III, comparable numbers of Mf were re-
262
RAO ET AL. TABLE I Effect of Iv on L,” Recovery in A. aegypti
Groupb
Hr*/days after treatment
I II
B. malayi
B. pahangi
Experimental
Control
Experimental
Control
2* 2*
0 0
5.88 2 4.24 6.20 k 3.26
8.61 * 1.46 6.42 2 2.13
I II
24* 24*
0 0
4.72 + 3.80 5.24 t 4.14
5.91 + 2.38 4.70 f 2.40
I II
7 I
0 0
3.78 k 3.20 4.10 2 3.80
5.82 k 2.52 6.14 ” 1.29
I II
15 15
0 0
5.16 2 3.80 5.83 f 3.58
0 0
5.28 + 2.32 5.89 2 2.12
I II
30 30
0 0
4.32 f 2.22 5.60 k 3.42
1.68 f 1.53 2.24 2 1.72
6.04 2 0.86 7.10 * 1.20
I II
60 60
0.28 k 0.42 0.32 f 0.50
6.14 f 2.10 5.18 2 1.4
3.2 f 1.60 4.4 2 2.12
5.82 f 3.14 6.27 2 1.56
I II
90 90
0.62 * 0.82 0.54 * 0.86
4.20 2 4.3 6.41 iz 3.6
4.6 2 5.2 5.4 2 3.2
1.28 2 3.24 6.18 ” 2.42
LINumber of L,‘s (mean f SE)/mosquito, 14 days after the blood meal. b Groups I and II represent duplicate experiments. Mosquitoes were fed on one treated or untreated infected jird. Fifty mosquitoes were dissected in each group.
covered from the abdomen of mosquitoes in both experimental and control groups. There was a decrease in the number of Mf recovered from the abdomen 72 hr after the blood meal and no further developmental stages of the parasite were recovered from mosquitoes fed on treated jirds. However, sausage stages were recovered from thoracic muscles of control mosquitoes 72 hr
after feeding on untreated jirds. Microfilariae recovered from the guts of mosquitoes fed on treated jirds were inactive and presumed dead. Effect of Iv treatment on Mf development in mosquitoes fed in vitro. To further evaluate the efficacy of Iv, mosquitoes were allowed to feed on drug-treated microfilaremic jird blood in vitro. Table IV shows that
TABLE II Effect of Iv on B. malayi L,‘s in A. aegypti“
Groupb
Hr*/days after treatment
I II
24* 24*
I II
7 I
Mf/20 (~1(mean f SE) Experimental
Number of L,‘s recovered (mean f SE)
Control
Experimental
Control
88 2 7 92 k 3
72 2 3 81 2 2
0 0
4.92 + 3.56 5.12 + 2.24
104 2 6 94 2 8
96 2 5 9024
0 0
8.14 + 2.86 7.86 * 4.20
0 Mosquitoes (n = 50) were dissected on the 6th day post blood meal. b Groups I and II represent duplicate experiments. Mosquitoes were fed on one treated or untreated infected jird. Mf, Microtilariae from drug treated (experimental) and untreated (control) jird blood.
IVERMECTIN
AND
TRANSMISSION
OF BRUGIAN
263
PARASITES
TABLE III Comparison of Intake of Microfilariae (MB and Sausage Stage (S) Development in A. aegypti” Fed on Iv-Treated B. malayi Infected Jird Hr after Infection
Experimental
Control
Group”
Head
Thorax
Abdomen
Head
Abdomen
24
I II
0 0
0 0
119 2 29(MD 88 2 16 (M~J
0 0
0 0
64kll 58 -t 8
48
I II
0 0
0 0
72 -t 10 (M~J 23 t 11 (M~J
0 0
0 0
36 + 12 (Mf) 42 + 8 (MD
72
I II
0 0
0 0
17 + 10 (Mf) 0
0 0
6 2 3 (S) 9 + 2 (S)
0.6 2 0.8 (Mt) 0
%
I II
0 0
0 0
0 0
0 0
7 + 3 (S) 10 + 2 (S)
2 k 6 (M~J 0.4 k 0.2 (Mt)
120
I II
0 0
0 0
0 0
0 0
8 + 4 (S) 6 + 5 (S)
0 0
ThOEWi
(Mf) (Mf)
y Fifty mosquitoes were dissected in each group. Mosquitoes were fed on one treated or untreated infected jird. b Groups I and II represent duplicate experiments.
L,‘s of B. malayi were not recovered from the experimental group even though comparable numbers of Mf were recovered from control and experimental groups of mosquitoes, 24 hr after the blood meal. Effect of Iv on lectin binding to Mf. The results in Table V show patterns of lectin binding to B. malayi and B. pahangi Mf recovered from Iv-treated and untreated jirds. These data indicate that Iv treatment causes surface membrane changes with new carbohydrate components such as Nacetyl galactosamine, D-mannose, and Lfucose, exposed on the surface in addition
to N-acetyl glucosamine. Very bright fluorescence was observed with FITC-WGA and -PWM, compared to Con A, HPA, and PSA. Lentil lectin, SBA, DBA, and UEA bound weakly to the sheath surface of Mf. The binding was completely inhibited by the competing sugars at 200 mM concentration. This pattern of altered carbohydrate complexities was observed on Mf recovered from jirds through 15 days after Iv treatment. Microfilariae recovered 30 days after treatment showed only N-acetyl glucosamine, similar to normal Mf from untreated jirds. Microfilariae preincubated
TABLE IV Effect of Iv” in in Vitro Feeding Experiments Using B. malayi Microtilariae
Number of mosquitoes dissected
Number of Mf recovered 24 hr post blood meal/mosquito
Number of L, recovered 14 days after blood meal/mosquito
Mosquitoes infected (%o) with L,
Experimental (treated): 110*2 I II 70 2 4
25 25
48 2 12 24 t- 8
0 0
-
Control (untreated): I 114 % 6 II 84 + 3
25 25
36 t IO 28 2 7
6.2 2 3.1 5.3 + 4.2
Groupb
Mf/20 ~1 of blood
68 76
a Iv (50 &ml) was added to jird blood containing B. malayi Mf for in vitro membrane feeding of mosquitoes. b Groups I and II are duplicate experiments. Results are represented as means 2 SE of larval stages.
264
RAO ET AL. TABLE V Effect of Iv on Lectin Binding Characteristics of B. malayi and B. pahangi Microfilariae Mf from jirds’
LectinsO WGA
Specificity
Con-A
N-Acetyl glucosamine N-Acetyl glucosamine o-Mannose
LCH
D-Mannose
PSA
D-Mannose
HPA
N-Acetyl galactosamine N-Acetyl galactosamine N-Acetyl galactosamine L-Fucose
PWM
SBA DBA UEA
Inhibitory sugarsb + + + + + + + + +
Treated
Untreated
Bm
BP
Bm
BP
+++ +++ ++ + ++ ++ + + + -
+++ +++ ++ + ++ ++ + + + -
+++ +++ -
+++ +++ -
-
-
-
-
-
-
-
-
-
-
-
-
a FITC-lectins were used at 100 &ml concentration. b Corresponding inhibitory sugars were added at 200 mM concentration. ’ Mf (Bm, B. mafuyi; Bp, B. pahangi) from jirds were isolated and tested for lectin binding activity 7 days after Iv treatment). Degree of Fluorescence: - , negative; + , weak; + + , bright; + + + , very bright.
with Iv in vitro, however, failed to show the additional carbohydrate moieties (data not shown).
1988). Ivermectin also appeared to reduce Mf counts of subperiodic B. malayi in leaf monkeys (Presbytis cristata) (Mak et al. 1988). Similarly, marked Mf reduction was DISCUSSION observed in Mastomys natalensis infected Ivermectin, a semisynthetic macrocyclic with B. malayi (G. Lammler and I. Sanger, lactone, has been shown to have activity as unpublished observations, quoted by Camp an antitilarial agent (Campbell 1985, 1987). bell, 1982). Ivermectin was, however, Several field studies involving Iv treatment found to be ineffective in reducing the Mf of of human subjects infected with On- B. pahangi in M. unguiculatus or Felis cachocerca volvulus suggested that Iv re- tus (Denham 1982). Studies with 0. volvuduces skin microfilaremia and thus inter- lus, Litomosoides carinii, and Acanrupts the transmission of the parasite to its thocheilonema viteae suggested that Iv ininsect vector (Cupp et al. 1986, 1989; terfered with embryogenesis and the Prod’hon ef al. 1987). Efficacy of Iv on Mf release of Mf from adult worms (Awadzi et of lymphatic filarial parasites is varied de- al. 1985; Schulz-Key et al. 1986; Zahner et pending upon the species. Previously, Iv al. 1987). In this study, Iv appeared to have was found to be completely effective in a long term effect on the subsequent develclearing the blood Mf of Wuchereria ban- opment of Mf in the mosquito vector. Becrofti within 5 to 12 days with reappearance cause the half life of Iv is between 2 and 8 of Mf after 3 months (Kumaraswami et al. days (Bennett et al. 1988; Chiu et al. 1986)
IVERMECTIN
AND TRANSMISSION OF BRUGIAN PARASITES
and is absorbed effectively into the adult worm tissues (R. E. Howells, unpublished, quoted by Campbell 1987) the drug would have sufficient time to act on an entire population of Mf. It is evident from the results of our study that Iv does not suppress the microtilaremia of B. malayi and B. pahangi in jirds for a period after treatment. Brugia malayi circulating Mf started decreasing only 30 days after treatment and B. pahangi Mf levels were unchanged. Interestingly, A. aegypti mosquitoes fed on microfilaremic jirds, 1530 days post-treatment never became infective. The ingested Mf from these treated animals failed to develop either to the sausage stage, L,‘s, or L,‘s. Thirty days after a single dose treatment B. pahangi mosquito infectivity was reestablished. Sixty and ninety days post-treatment, the number of B. pahangi L,‘s recovered from the experimental group of mosquitoes and the percentage of infected mosquitoes were comparable to controls. In contrast, mosquitoes fed on B. malayi jirds showed comparatively fewer L,‘s even at 60 and 90 days post-treatment and only a small percentage of mosquitoes were infected. The results of in vitro feeding experiments further corroborated these in vivo findings by demonstrating the failure of Mf to develop to L,‘s. These results support previously reported observations that B. pahangi infections in cats or Meriones are not susceptible to Iv (Denham 1982) compared to B. malayi infections in M. natalensis (Campbell 1982). Correspondingly lesser effects on the infectivity of mosquitoes fed on Iv-treated B. pahangi jirds were observed in the present study. In addition to the ability of Iv to reduce circulating microtilaremia, as reported earlier, an alternative mechanism of Iv activity is suggested by the results of the present study, i.e., biochemical alteration of the Mf surface. Such changes have been previously suggested to affect Mf development in the insect vector (Furman et al. 1983;
265
Devaney 1985). We found changes in the surface lectin binding properties of Mf recovered from jirds through 15 days posttreatment. Additional sugar moieties like Dmannose, N-acetyl galactosamine, and Lfucose were identified on the sheath surface of Mf exposed to Iv in vivo. In contrast, the present and previously reported studies with in vitro incubations of Mf with Iv and or DEC (Nwachukwu et al. 1987; Devaney 1985; Rao et al. 1987a, b) failed to demonstrate changes in lectin binding properties of Mf. It may be possible that our observations regarding the in vivo activity of Iv are the results of synergistic activities of host components with the drug. Further studies to elucidate in vivo drug interactions are being conducted to extend our knowledge about the transmission blocking activity of Iv. ACKNOWLEDGMENTS We thank Dr. Charles H. Courtney, Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville, Florida for providing us a sample of Ivomec and Mrs. Robin Buck for excellent technical assistance. U.R.R. was supported by funds from the Filariasis component of the UNDP/ World Bank/WHO special program for Research and Training in Tropical Diseases. The study was supported in part by research Grant AI 20052 awarded by the NIAID of the NIH. REFERENCES AWADZI, K., DADZIE, K. Y., SCHULZ-KEY, H., HADDOCK, R. W., GILLES, H. M., AND AZIZ, M. A.
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1989; accepted with revision
