BIOCHEMICAL AND IMMUNOCHEMICAL EFFECTS OF CERTAIN ...ir.amu.ac.in/7353/1/DS 2495.pdf · Tabel -5 Effect of anthelmintics on membrane bound marker enzymes of G. explanatum ( soluble

BIOCHEMICAL AND IMMUNOCHEMICAL EFFECTS OF CERTAIN ANTHELMINTICS ON

SOME GASTROINTESTINAL HELMINTHS

DISSERTATION SUBMITTED FOR THE DEGREE OF

MUittV of $I|tlQS[QpI)P P A. ®T^. IN

ZOOLOGY

*v,-'

BY

GUbSHAN ZIA

SECTION OF PARASITOLOGY DEPARTMENT OF ZOOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

1994

ii

I }

DS2495

J^^Suti\ifii''

$ ?C*>>~v>'

J</c»»*<; Aligarh Muslim University

Section of Parasitology, Department of Zoology A M U . , Aligarh - 202 002 [INDIA]

( J ) . (0571) 401647/400922, Ext, 302

Wajih A. Nizami

Dated A?.:^.:.^"^."^^

This is to certify that the dissertation entitled

"Biochemical and immunochemical effects of certain anthelmintics

on some gastrointestinal helminths" submitted by Ms Gulshan Zia,

embodies original work done by the candidate herself. The entire

work, was carried out under my supervision and that I allow her to

submit the same in partial fulfilment of the reqiilromnntn for the

degree of Master of Philosophy in Zoology of this University.

Prof. W.A. Nitimi

(Supervisor)

CONTENTS

Page

List of Tables ;

List of Figures 11

List of Plates ijl

AcJcnowledgemenLs JV

CHAPTER I INTRODUCTION ]

CHAPTER II HISTORICAL REVIEW 9

CHAPTER III MATERIALS AND METHODS 31

CHAPTER IV RESULTS ^

CHAPTER V DISCUSSION 55

CHAPTER VI SUMMARY Q 3

BIBLIOGRAPHY 66

LIST OF TABLES

Table -1 Enrichment of surface plasma membrane enzyme activities in comparison with fresh worm homogenate of G.explanaturn.

Table -2 Enrichment of surface plasam membrane enzyme activities in comparison with fresh worm homogenate of G. crumenifer.

Table -3 Effect of anthelmintics on membrane bound marker enzymes of G. crumenifer ( soluble fraction).

Table -4 Effect of anthelmintics on membrane bound marker enzymes of G. crumenifer ( membrane fraction).

Tabel -5 Effect of anthelmintics on membrane bound marker enzymes of G. explanatum ( soluble fraction).

Table -6 Effect of anthelmintics on membrane bound marker enzymes of G. explanatum. ( membrane fraction).

Table -7 Effect of some anthelmintics on in vitro 3

uptake of -glucose in G. crumenifer.

Table -8 Effect of oxyclozanide on the antibody titer in rabbit.

Page

47

48

50

51

52

53

56

63

(0

LIST OF FIGURES.

Fig 1. Flow diagram for the isolation of surface plasma membrane of amphistomes.

Fig 2. Chemical structures of the anthelmintic drugs used.

Fig 3. Effect of mebendazole on in vitro uptake of 3 H-glucose by G^ crumenifer.

Fig 5, Effect of Rafoxanide on in Yi.tro uptake of 3 H-glucose by G. crumenifer.

C'l)

Page

32

36

57

Fig 4. Effect of oxyclozanide on in vitro uptake of 58 3 H-glucose by G. crumenifer.

59 Fig 6. Diagrammatic representation of precipitation

peaks following reversed rocket immune- co electrophoresis.

LIST OP PLATES

PEiATE 1. Infected liver and rumen showing Giqantocotyle explanatum and Gaatrothylax crumenifer infections respectively.

PEJATE 2. Whole mount preparations of Gastrothvlax crumenifer and Giqantocotyle explanatum.

PLATE 3. Trasmission electron micrographs of Gastrothylax crumenifer and Giqantocotyle explanatum surface plasma membranes isolated after 20 min of detergent treatment.

PLATE 4. Transmission electron micrographs of surface plasma membranes of Gastrothylax crumenifer showing trilaminate structure, puffed membranes and pores.

Plate 5. Reversed rocJcet Immunoelectrophoresis of control and experimental sera against partially purified membrane antigens of Gastrothylax crumenifer.

Page

7

6

AA

A5

61

(iii)

ABBREVIATIONS USED

ATP CaCl

2 cm DDW DMSO g h

HBSS KH PO

2 4 M mA MBZ MOS min mm mM N nm nmole OXCL Pi RFZ Sec TCA ugm ul v/v w/v

Adenosine Triphc phate Calcium Chloride

Centimetef Double Diattiled Water Dimetnyi sulfoxide Gram Hour Hank's Balanced Salt Solution Potassium dihydrogen phosphate

Molar Milliampere Mebendazole Membrane disrupting solution Minute Mill meter Milli molar Normal Nano meter Nano mole Oxyclozanide Phosphorus Rafoxanide Second Trichloroacetic acid Micro gram Microliter volume/volume Weight/Volume

ACKNOWLEDGEMENTS

I fall short of words to express my gratitude to ray-

supervisor Professor W.A. Nizami for his inspiring guidance,

encouragement and keen interest throughout the course of this

study. Infact it was a great previlage to work under his

guidance.

Sincere thanks are due to former chairman Professor M.M.

Agarawal, present chairman Professor A.K. Jafri, department of

Zoology, A.M.U. for providing the necessary laboratory

facilities.

I wish to exress my heartfelt gratitude to my parents for

their love, encouragement and moral support.

Special thanks to my colleagues Dr. S.M.A. Abidi, Dr.Parveen

Khan, Dr. Malik Irshadullah, Mr M. Khalid Saifullah , Mr Afsar

Ali, Mr Gul Ahmad and Ms Nazneen B. Khan for providing help

during the tenure of this work.

I also record my sincere thanks to my friends who never left

me alone with my problems.

I am thankful to Mr Mirza Rais Baig for typing this

manuscript and Mr Kalam & Mr Samiuddin for collecting the

parasites.

Finally I am grateful to A.M.U. Aligarh for providing the

financial assistance.

( Gulshan Zia)

(iv)

1

INTRODUCTTION

Arophiatomes are thick bodied conical or cylindrical

digeneans, distinguished from other forms by posteriorly located

acetabulum, responsible for firm attachment to the host tissue.

Infection of amphistomes are commonly found in sheep, goats,

cattle and water buffaloes in India as well as in other tropical

countries. The disease amphistomiasis is caused by the massive

infection of immature paramphistomes and this disease is

characterized by acute gastro-enteritis with high morbidity and

mortality rates, particularly in young stock. Rumen parasites

generally render a low pathogenicity causing acute catarrhal and

haemorrhagic inflammation in the abomasum, duodenum, and jejunum

with associated anaemia, hypoproteinaemia. While the amphistomes

parasitizing the bile duct cause haemorrhage, pronounced

periductal fibrosis and other hyperplastic changes (Kulasiri and

Seneviratne, 1956; Arora and Kalra, 1971; Jha et al., 1977),

Migrating immature paramphistomes in intestine cause severe

pathological changes characterized by general weakness,

increasing anorexia and polydypsia.

In a survey from our laboratory on the epidemiology of

amphistomiasis 71.4* of buffaloe's rumen were found infected with

different species of amphistomes with varying intensities of

Gastro' yla; crumenifer (32.7%), Fischoederius elongatus (6.0*),

Paramphistomum epic1itum (51.9 X), Qrthocoelium scollpcoellum

(16.IX). While in liver, Gigantocotyle explanatum infection was

observed in 19.6\ animals which was most widespread amphistomes

of bile duct (Mattison et al., 1994).

India is basically an agricultural country, where livestock

contribute considerably and provide 3 2* of the total energy

requirement of rural areas (Odend'hal, 1972). Acoording to FAO

(1986) production report asian countries have 96* of the world's

buffalo population, of which India has the largest population

(54\) of buffaloes. In 1985, total yield of milk and meat from

India was 21.391 and 0.139 million tonnes, while 32 and 1.0

million tonnes respectively were produced by the remaining world.

Besides dairy products, buffaloes form the main sources for

various agro based industries like milk, meat leather industries,

transport as well as serve as a source of fuel and fertilizers.

It has been estimated that the animal husbandry generated

1,08,640 million in 1984-85 which is roughly 18* of the total

agricultural out put. The dairy products are an important source

of nourishment to our diet. Buffaloes contributes 58* of the

milk though they form only 30* of the total bovine (Cattle and

buffaloes) population (Acharya, 1988).

Despite their importance in the country's economy the net

contribution made by animal wealth is not very satisfactory. Poor

animal health has been considered as one of the main reason for

meagre output. Besides viral, bacterial and protozoan infections,

a bewildering array of helminth parasite cause heavy morbidity

and also mortality in livestock during epidemic out break. In

view of losses incurred by such parasitic iniections, it is

esrsential that we should protect our livestock from these

infections. It has been suggested that the milk and meat

production and draught animal power can be imporved through

selective breeding and effective animal management. However,

desired results can only be obtained if equal attention is paid

to the health of animals.

In order to control any parasitic infection it is essential

to have a complete background of parasite metabolism. Most of

the physiological information on the adult trematodes has come

from observation on the functional morphology of the various

organ system like tegument, sucker, alimentary canal,

parenechyma, excretory system, reproductive system etc.

The tegument of trematodes consists of an outer anucleated

syncytial layer which is contributed by the inner nucleated

layer comprising the tegumental cells and other cellular

organelles. It is externally covered by plasma membrane which is

involved in many physiological processes including the absorption

of nutrients, excretory processes and immunological interactions.

In addition to this, surface plasma membranes are also involved

in protection from host digestive enzymes, immunoglobulin attack,

and transport of a large number of solutes by a variety of

absorption mechanisms (Diffusion, active transport, pinocytosis

etc). The tegument of helminths is known to be covered with an

unstirred layer which includes the glycocalyx. It is largely

composed of glycoprotein, with projecting side chains of

oligosaccharides and gangliosides, uoth bearing terminal sialic

and other acid residues. The various ionic components of host

origin are absorbed to the glycocalyx by electrostatic

t

interaction. Further, the glycocalyx may act as "cation exchange

reain" by concentrating those cations which are necessary to

maintain maximum enzyme activity (Lumsden, 1972).

Therefore, the tegumental surface of the trematodes form an

interface vMith the habitat and it is site for a variety of

biochemical processes like absorption, digestion, "protection"

and "infromation transfer". Each of these processes play a vital

role in maintaining the parasitic mode of life of these

organisms. Knowledge of the surface plasma membranes is therefore

fundamental to understand the host- parasite relationship, which

provides basic information for the development of any effective

control programme through immunological or pharmacological means.

Those drugs that cause dysfunction at the surface should be

considered highly sucessesful therapeutic agents.

Anthelmintic treatment for the control of immature and

immature paramphistomiasis has been practised at various levels.

The removal of adult infections is probably of little direct

benefits to the animal but may be prophylactic, as it serves to

reduce the reservoir infection for intermediate host. Several

anthelmintics have been used against immature stages in sheep and

goat and occassionally proved effective in the field. However, no

drug is available showing high efficacy against adult flukes and

the availability of highly effective and safe drugs for the

treatment of immature worms are very limited. Various drugs have

been tested against these infections by a number of workers

(Ahmad, 1984 and Boray, 1986) and suggested that the mode of

action of anthelmintics is generally associated with energy

yielding processes, nutrient upta)ce mechanism, and neuromuscular

coordination.

Although, considerable progress has been made in recent

years on the chemotherapeutic effect of various anthelmintics and

their mode of action but very little is known about their

influence on specific immune status of the treated host. Such

information will generate indirect evidence on effectiveness of

anthelmintics.

As mentioned earlier, the control of parasitic diseases

relies largely on prophylactic or therapeutic application of

antiparasitic drugs. In many instances these measures are only

partially effective and lead to the selection of drug resistance

within the parasite population. Therefore chemotherapy required a

rational approach rather than emperical approach. Besides this,

drugs have appreciable with holding capacity in biological system

and produce profound toxic effects which is an important aspect

and can not be ignored.

Available literature on this aspect clearly reveal that

anthelmintic drugs affect the immune system of the host. It has

been reported that antiparasitic compounds affect the lymphoid

organs, T and B cells, lymphocyte count, as well as bone marrow

progenitor cells. Besides this, the antibody titer is also

affected. (Von Behren et al., 1983 ; Goldstein et al., 1969 and

Thigpen et al., 1975). Such immunomodulatory action of

anthelmintics may result in impairement of host immune system,

leading to increased susceptibility to subsequent infections and

loss of resistance to parasitic infection.

0

study of the relationship between the immune system and

chemotherapy in parasitic diseases is still relatively

unexplored. Many of the anthelmintic agents currently in use are

Icnown to require an intact immune system to eliminate the

parasites from the host. To date, combined immunostimulation and

chemotherapy will only more effective than chemotherapy alone for

helminth parasite treatment.

In view of the above facts, the present study was undertaken

to investigate the molecular action of some Icnown anthelmintics

on the surface plasma membrane bound enzymes. Since the surface

plasma membrene seems to be primary target for the drug action 3

and involve in trasmembranosis, therefore H-glucose uptake was

analysed in presence and absence of drugs. In addition to this,

the effect of oxyclozanide on the antibody titer against the

membrane antigen was also investigated by using reversed rocket

Immunoelectrophoresis.

It is expected that the outcome of this study will

definitely contribute in designing the pharmacological control

of these economically important group of parasites. Further, this

study will provide some data relevant to the immunoprotection

studies for an epidemic area of amphistomiasis.

Plate ]: Infected liver and rumen sho wing a: Heavy infection of liver amphistome Gigantocotyle exElanatum. Bile duct (arrow heads) enlarged and extensive liver damage can be seen. b: Heavy infection of Gastrothylax crumenifer in the anterior dorsal sac of the rumen of buffalo. The flukes often occur in clamps (arrow). Mucosa at the attachment site becomes depigmented.

Plate 2: Whole mount preparation of a: Gagtrothylax crumenifer t): Gigantocotyle explanatura staintd with alcoholic borax carmine (scale bar 2 urn). Ph: Pharynx, U: Uterus, G: Gut, VP: Ventral pouch, Te; Testes. Ov: ovarv: Ovarv. AC: Acetabulum.

HISTORICAL REVIEW

Parasites pose a continual and unacceptable threat to the

well being of human being and to the farm animals all over the

world. The cost of parasites in terms of human misery is

incalculable,, although various reports are available on economic

loss from our farm animals. Parasites have evolved ways of

surviving in nutritionally rich, but immunologically hostile

environment of their host. These adaptations that make them

unique and fascinating organisms to study. Parasites generally

display a combination of biological and chemical adaptations

unique in animal world and at the same time display a range of

methods of evadingthe host immune response in order to establish

a delicate and harmonious host- parasite relationship.

In view of the clinical and economic importance of these

parasites, eradication of parasites has been the goal of various

nationnl and international organizations. Control of parasites of

livestock usually aims at reducing the parasitism to a level

having no effect on productivity provided that it is economically

feasible. For this, different methods of control have been

adopted by the parasitologists.Chemotherapeutic and immunological

control have received considerable attention during the recent

past. In the former approach a number of new compounds have been

synthesiser an screened for their efficacy. The available

literature indicates that in most of the cases the mode of action

is not completely known or if partially known then its

toxicological aspects have not been investigated. The main aim of

10

pharmacologists is to control the parasites either at individual

or mass level. Today, various antiparasitic compounds are

available but the effect of these compounds on the host

particularly at immunological level is more or less neglected.

Parasite chemotherapy has attracted considerable attention

over the past two decades and numerous reports on drug

development, their mode of action and laboratory and field

trials are available. Comparative studies on various drug

formulation, their chemistry and the selective toxicity have also

been investigated. In 1909 Paul Ehrlich laid down the foundation

of chemotherapy of parasites and proposed that the inhibition of

enzymes that were crucial to the parasites but not to the host

might be the basis of a rational approach to the chemotherapy of

parasites.

Von Brand (1973) suggested that most drugs interfere with

the enzyme systems by inhibiting them and thus interfere with the

metabolic processes. Such studies would be of immense value in

knowing the effect of anthelmintic drugs at molecular level in

the helminth parasites. Carbohydrate plays a major role in energy

metabolism of helminths, which require a continuous supply of

energy (ATP), to motivate the complex metabolic processes

essential for their normal growth and development. Hence,

carbohydrate metabolism has been extensivly studied in helminths.

Since then many compounds have been screened for their

chemotherapeutic effects and the available literature has been

comprehensively reviewed by many workers ( Bando, 1951; Guilhon,

1968; Oakley, 1978; Van den Bossche,1978, 1980; Mansour, 1979;

l l

Armour, 1983; Coles, 1983; Gutteridge, 1985; Bogan and Armour,

1986; Boray, 1986; Campbell, 1986 a; Prichard, 1978 , 1986;

Chappell, 1988; Subrahmanyam, 1987). Moreover many books such as

Chemotherapy of Parasitic Diseases ( Campbell and Rew, 1986),

Chemotherapy of Gastrointestinal Helminths ( Van den Bossche et

al., 1985 ) have been published and reviewed the available

literature.

Among the various compounds that have been tested so far,

the benzimidazoles, salicylanilides and organophosphorous

compounds are found to be quite effective against helminth

parasites. In the present work three anthelmintics were selected

namely mebendazole from the benzimidazole group, oxyclozanide and

rafoxanide representing the salicylanilide group.

BENZIMIDAZOLE COMPOUNDS: The benzimidazoles were introduced as a

group of safe and effective broad spectrum anthelmintics with the

discovery of thiabendazole by the Merck group in 1967. Further

Smith Kline and French introduced fenbendazole in 1967 and since

then many other international pharmaceutical companies have

brought a number of benzimidazole compounds like cambendazole,

mebendazole, oxibendazole, thiophanate, fenbendazole, albendazole

and oxfendazole on to the market.

MEBENDAZOLE: Mebendezole ( Methyl - 5 - benzoyl benzimidazole -2-

carbamate) was introduced as a new potent anthelmintic compound

by Janssen Pharmaceutica in 1971 as product No R 17635. In vitro

efficacy and therapeutic action of mebendezole against a number

of helminths was reported by varic is workers ( Chaia and Cunha,

1 1

1971; Brugmana et al., 1971 and Banerjee et al.,1971). Van den

Boasche (1972) reported for the first time the biochemical effect

of mebendazole on a number of nematodes and cestodes both in in

vitro and in vivo conditions. A number of biochemical effects of

this drug have been demonstrated in different species of

helminths which together account for its broad spectrum

anthelmintic efficacy. De Nollin and Van den Bossche (1973) have

studied biochemical effects of mebendazole on Trichinella

spiralis larvae in vitro and reported that mebendazole inhibits

the uptake and /transport of glucose and increases glycogen

depletion in larvae. This drug also reduces the uptake of

fructose 3-O-methyl glucose, glycine, methionine, proline and

palmitic acid by Ascaris suum (Vanden Bossche and De Nollin,

1973). Recently Ahmad and Nizami (1991) has studied the effect of

mebendazole on the carbohydrate metabolism of Gigantocotyle

explanatum under in vitro conditions and concluded that

mebendazole inhibits the glucose uptake due to the inhibition of

phosphomonoesterases and the rapid depletion in endogenous

glycogen content may be due to stimulation of phosphorylase and

phosphoglucomutase.

Mebendazole induces many other effects on energy metabolism

in helminths like malate induced phosphorylation in Ascaris

mitochondria (Van den Bossche,1972) in viyo and in vitro decrease

of ATP synthesis and turnover of nucleotides (Rahman and Bryant,

1977; Bryant et al.,l976). A number of reports indicate that

mebendazole binds to mammmalian tubulin , the constituent protein

of microtubules (Kohler and Bachmann, 198 0, 1981; Ireland' et

13

al.,1982). Kohler and Bachmann (1981) demonstrated that this drug

could inhibit ( H) colchicine binding to nematode tubulin in

competitive manner. Inhibition of microtubule system might affect

motility, absorption, secretion and enzyme activity in parasites.

Further,invitro inhibition of acid and alkaline phosphatses,

glucose-6-phosphatase and 5' nucleotidase was reported due to

mebendazole in Avitellina lahorea (Ahmad and* Nizami,1987).

Mebendazole has no effect on hexokinase activity of the helminths

while phosphorylase and phosphoglucomutase are affected by this

drug, which ultimately causes the depletion in glycogen content

and inhibits glucose uptake (Van den Bossche, 1972; Ahmad and

Nizami, 1987).

Biochemical changes caused by mebendazole in helminth

parasites have also been investigated histochemically in

order to support biochemical studies. Progressive time related

morphological changes in cestodes and nematodes after treatment

of the host with mebendazole have been demonstrated by electron

micrscopy ( Borgers et al.,1975; Verheyen et al., 1976).

Ultrastructural changes in the encysted Trichinellla larvae,

during and after treatment of the rat with mebendazole have been

reported (De Nollin et al., 1974). Degenerative changes were also

observed in the immediate infected host muscles like oedematous

swelling and intracellular vacuolization while loss of glycogen

was found in larvae cells. Borgers and Verheyen (1976) and

Verheyen et al. , (1976) have also observed tegujient. . changes in

Hymenolepis. nana and Taenia, taeniformis due to drug action and

suggested that disrurMon of microtubule formation may pre snt

the outward movement of secretions from the subtegument to the

apical tegument.

Besides these effects in vitro trials of mebendazole have

also shown inhibitory action in the egg development, increase in

the rate of egg out put (Banerjee et al.,1971; Banerjee and

Prakash, 1972; Ahmad et al., 1987), inhibition of the intestinal

secretions (Atkinson et al. , 1980) and inhibition of acetyl-

cholinestrase secretion In helminths (Watts et al., 1982).

Further, mebendazole is also reported to reduce the rate of egg

production in benzimidazole resistant nematodes at normal

therapeutic doses (Kelly et al., 1975). Many field trials and in

vivo experiments have revealed that mebendazole has a broad

spectrum effects against helminth parasites in both domestic and

wild animals. Fernando and Denham (1976) have studied the

efficacy of mebendazole under i,n vivo conditions against encysted

muscle larvae of Trj.chinella spiralis in mice while Kelly et

al.(1975) have reported the effect of particle size on

anthelmintic efficacy of mebendazole against Nippostongylus

brasiliensis in the rat.

SALIC}YLANILIDES: Among the salicylanilides " Diaphene" and

"Hilomid" were possibly the first compounds used as flukicides

(Boray et al.,1965; Lienart, 1963;). Salicylanilides and

substituted phenols show variable activity against helminths, but

are usually most active against blood sucking worms ( Stampa and

ferbi nche, 1961; Horak, 1962; 1964; Boray, 1969; Sinclair and

Prichard, 1975). These compounds generally bind with the plasma

proteins and cause metabolic disorders in the parasites. Lee

15

(1973) reported that fasciolicidal activity of salicylanilides in

sheep was dependent on the extent to which they persist in the

plasma. Salicylanilides and substituted phenols are also potent

uncouplers of oxidative phosphorylation (Williamson and Metcalf,

1969). Scheibel et al.(1968) demonstrated uncoupling of oxidative

phosphorylation in tape worm due to niclosamide under in vitro

conditions. Oxyclozanide, nitroxynil, rafoxanide and nitroscanate

uncouple oxidative phosphorylation in F. hepatica and A^

lumbricoides (Corbett and Goose, 1971; Van den Bossche, 1972 and

Cornish and Bryant, 1976). Further, Cornish et aO,. (1977) and

Prichard (1978b) suggested evidence of uncoupling during in vivo

trials.

OXYCLOZANIDE: Oxyclozanide is one of the most potent

salicylanilide compound active against trematodes and cestodes.

Broome and Jones (1966) have reported the therapeutic activity of

oxyclozanide against Fasciola sp. They found that this drug is

absorbed and binds to plasma proteins and transformed in liver to

the anthelmintically active glucuronide and concentrated in bile

in the environment of adult fluke.

A number of successful trials have been carried out on the

efficacy of this drug against F. hegatica, H. diminuta, H.

microstoma. Stilesia hepatica and few amphistomes (Broome and

Jones 1966; Wally, 1966; Harrow; 1969; Foreyt and Todd, 1973;

Hopkins et al., 1973; Corba et al., 1976; Georgiev and Gruev,

1979; Denev et al., 1985). Some in vitro studies have also been

accomplished on the biochemical and metabolic effects, resulting

into the inhibition of malate dehydrogenase o*. F, hepatica , F.

qlqantlca, Fagciolopais bualci, and Paramphistomum explanatum. (

Lwin & Probert, 1975; Coles et al., 1980 and Probert et al.,

1981) and uncoupling oxidative phosphorylation (Corbett & Goose.

1971). Further, Edwards et al., (1981) also reported the effect

of oxyclozanide on the metabolism of F. hepatica.

Further reports are also available on the efficacy of

oxyclozanide alone or in combination with levaraisole against both

mature and immature paramphistomes (Schielhorn Van veen and

Bida,1975; Chhabra et al.,1977; Prasad, 1983). Oxyclozanide has

been formed highly effective against the pathogenic immature

forms in sheep or goats at a dose rate of 15 mg /Kg body weight.

Many reports from Eastern Europe and from USSR confirm the

efficacy of the drug against the adult parasites in both sheep

and cattle ( Denev et al, 1985). It appears that in an outbreak

of acute paramphistomiasis in calves oxyclozanide may be the most

suitable drug.

RAFOXANIDE: This salicylanilide is highly effective against adult

and to a lesser extent against immature F. hepatica (Lucas, 1967;

Boray and Happich, 1968; Ross, 1970; Armour and Corba, 1970;

Annen et al., 1973; Boray et al^ 1973; De Keyser, 1980). This

drug has been found active against some nematodes particularly i

Haemonchus contortus and nasal mj asis of sheep Oestrus ovis

(Lucas, 1967; D'souza et al,. , 1992) Increased dose rate from the

normally recommended was found highly active against fluke aged 4

weeks or older (Armour and Corba, 1970; L wards and Parvy, 1972).

Fasciolicidal activity of the rafoxanide is due to retention

ability in F asma (Lee, 1973). The half life is about 4 days and

17

it binds to plasma proteins with a high affinity. Cornish et al.

(1977) and Prichard (1978b) provided the evidence of uncoupling

of oxidative phosphorylation in vivo. Some i,n vitro studies have

also been accomplished on the biochemical and metabolic effects,

resulting in the inhibition of succinate dehydrogenase and

fumarate reductase system in F. hepatica ( Duwel and Metzger,

1973; Metzger and Duwel, 1973; Coles and East,1974).

The foregoing review of the literature, indicates that all

the above mentioned compounds show anthelmintic activity in one

way or the other. Different parameters have been used by the

various investigators for the assessment of their efficacy. In

vitro studies such as biochemical effects, ultra structural

damages and electrophysiological changes on the surface of the

parasites also provide informations about the mode of action of

a particular drug which ultimately forms the basis of in vitro

trials.

In recent years the electron microscopic evaluation of

damage have also been used as a powerful tool to study the actual

site of morphological disorganization due to drug action.

Erasmus (1970) pointed out through scanning electron microscopy

the physiological importance of the tegumental surface of the

digenetic trematodes in the host parasite relationship. Recently,

Dunn et al. (1987) and Mattison et al. (1992) have reported some

specialized structures of tegument of the amphistomes under study

and suggested their possible role in establishment of parasitic

mode of life and possible chemoreceptive function. Drug induced

topographical changes have been reported by Probert et al. (1982

1 ••]

in Moniezla. expansa treated with oxfendazole and Praziquantel in

vitro. Ahmad et al. (1987) have also reported some topographical

changes and observed that oxyclozanide produce most pronounced

damage in G. explanatum while mebendazole produced least effect.

Pax et al. (1978) have reported that praziquantel causes

contractions in the musculature of S. mansoni and showed that

this drug decreased the influx of Na and Ca . They suggested that

spastic paralysis In the worms may be related to depolarization

of the cells, elicited through inhibition of Na"- K* ATPase

function. Coles (1979) suggested that in Schistosoma.mansoni

praziquantel opens pores in membranes and permits a rapid influx

of Ca*"*" either directly or indirectly through an effect on the

influx of Nat

Mehlhorn et al. (1983) have also investigated the effect of

praziquaentel on some human trematodes namely Clpnorchis sinensis

.Metaqonimug yokoqawai, Qpisthgrchis viyerrlni. Paragonimus

westermani and S. japonicum and reported that all flukes exposed

to the drug contracted and showed tegumental alterations such as

papillae and smoothing of surface. In vivo effects of amasconate

have been studied by Voge and Bueding (1980) and reported

tegumental swelling in S. mansoni. Kohn et al. (1982) reported in

vivo effects of oxamniquine on the tegument of male S. mansoni

and found bubble like lesions.

Ultrastructural studies on helminths have revealed that the

tegument is metabolically active and plays an important role in

transmembranosis of sugars, amino acids and other substances (see

reviews by Lumsden, 1975 ,- Erasmus, 1977; Threadgold, 1984). The use

13

of radiolabelled subifc'ates in physiological studies of helmintHs

has provided a conclusive evidence that tegument play a vital

role in uptake mechanism. Further, by the use of this technique,

it is also possible to work out actual mode of uptake, site of

incorporation in the organelles or in the complex biochemical

moities (Hanna, 1975, 1976, 1980; Specian and Lumsden,1981)

The autoradiographic examination of labelled hexoses and

amino acid incorporation shows that a significant amount of

radioactivity incorporated into macromolecules in the tegument

and various other organs of trematodes. Monosaccharide absorption

has been studied mainly in F. hepatica and Schistosoma spp. and

the evidence suggest that hexose uptake occurs through the

tegument rather than oral orifices as gut involvement was

precluded by ligation of the pharynx (Mansour, 1959; Isseroff and

Read, 1974) or by the exclusion of the mouth from the test medium

(Roger and Bueding, 1975). Similarly Parkening and Johnson (1969)

have also observed the absorption of tritiated glucose by the

tegument and not by the intestinal caeca in starved or unstarved

adult Haemonchus medioplexus and Gorgoderlna spp. Recently Khan

(1991) have studied glucose transport kinetics in gut ligated

amphistomes under study and reorted that uptake of ^H-glucose

being taken up through the tegument.

In addition to this, a number of known drugs affect the

motility of the worms. Such motility recording , under in vitro

conditions, seems to be an effect! • e, quick and economic method

for screening a large number of drugs. Prob^rt et al.(198l)

studied visually the motality of the worms in Vitro in presence

. 1 )

of five flukicides including oxyclozanide in three trematodes of

veterinary importance namely F. gigantlca, Fasciolopglg buski and

Paramphistomum explanatum. Another drug diarafenetide also caused

paralysis in F. hepatica as observed visually by Rew et

al.(1983). Oxyclozanide is the only drug which has been tested

for amphistomes, while other drugs under study, have been

completely neglected. Recently, Ahmad and Nizami (1990) have

examined the in vitro effect of certain anthelmintics on the

motility of G. explanatum. It was observed that mebendazole

induces irreversible spastic paralysis, where as fenbendazole

causes irreversible disturbed activity. Metrifonate produced

rapid decrease in muscle tone leading to flaccid paralysis.

Oxyclozanide also induced immediate suppression of motility with

increasd muscle tone leading to irreversible spastic paralysis.

Further Ahmad and Nizami (1992) reported that oxyclozanide

induces time dependent irreversible spastic paralysis in four

amphistomes including G. crumenifer.

It is now an established fact that when an infected animal

is treated with anthelmintic drugs through any route of

administration, the drug molecules first come in contact with the

surface of the parasite. There after binding with an appropriate

receptor, the drug molecule are trasferred from the surface to

inside the cell and produce either morphological or biochemical

effects. This shows that parasite surface is a primary site for

drug action. With this assumption the present study was designed

to examine the effect of some known anthelmintics on the surface

plasma membranes of the amphistomes. Any adverse effect on the

11 biochemical configuration of the plasma membranes would certainly

influence the host-parasite relationship, which is the most

delicate and harmonious relationship in the successful

establishment of the parasite. Any disruption in this

relationship could be exploited for the control of infection.

This seems to be an important and appropriate rational approach

for the parasite control. ^

However, the other aspects of chemotherapeutic control like

toxic or side effects can not be ignored. Many pharmacological

agents induce a number of side effects and among these

immunotoxic effects are of prime importance.

IMMUNOPHARMACX>LOGICAL STUDIES

The main role of the immune system is to sustain the host

defence mechanism and to maintain homeostasis of the host.Two

main categories of unwanted consequences are produced by the

interaction of pharmaceutical drugs and the immune system (i)

alterations of host defence mechanism with either impaired or

enhanced normal immune response (ii) induction of qualitatively

abnormal immune response. The former effect is likely to

represent a direct toxic effect on the immune system while the

latter is associated with clinically significant unwanted

reactio-ns. Unfortunately immunotoxicologists largely focus their

attention on the immunodepressive activities, however immune

enhancement or immunestimulation should also be given equal

emphasis.

There are few scattered studies which have been carried out

on the chemotherapy of parasite infections and its relationship

22

with the immune status of the host. Since the disciplines of

chemotherapy and immunology have developed independently and

phrmacologists have mainly focusssed their attention on greater

parasiticidal properties in the drug development rather than

their interaction with the immune response, therefore an inter­

disciplinary approach is required to achieve this goal. Targett

(1985) (Comprehensively reviewed the available literature on

chemotherapy and immune response to parasitic infections and

grouped the various reports in following four categories:

(1) Immune dependence of antiparasitic chemotherapy

(2) Immunodepression by chemotherapy

(3) Chemotherapy and induction of immunity

(4) Immunopotentiation and chemotherapy

The only comprehensive review available on the immunotoxic

effects of drugs is by Descotes (1988). There are various anti

parasitic agents which are currently used as therapeutic or

prophylactic agents in order to control parasitic diseases which

aa/ersely affect the immune system leading to susceptibilty for

subsequent infections. Unfortunately, most studies of this nature

have been carried out on protozoans while helminths remained some

what neglected. The toxicity of the drugs is measured by

examining both cellular and humoral immunity. The various

parameters which have been used to test the toxicity have been

reviewed by Berlin et al.(1987) and Luster et al. (1982) and can

be grouped into three major categories:

Hi Histopathology of immunocompetent organs includes lymphoid

organs, thymus and various lymph nodes. Histopathology in

Z3

broad sense may include T and B lymphocytes, bone marrow cells

and their differentiation from a common multipotent stem cell.

The various aspects can be assessed by colony forming test or

Immunocytochemistry.

(2) Humoral immunity is another aspect to assess the immune

system which includes the level of immunoglobulins mainly IgG and

IgM using different immunoanalytical procedures like ELISA,

immunoblotting, lEP etc. Besides Igs level, haemagglutination,

plaque forming cell technique have also been used.

(3) Cell Mediated Immmunity. This can be assessed by classical

delayed type hyper sensitivity response to antigen, migration

inhibition test, lymphocyte proliferation assay using various

mitogen and lymphocyte toxicity.

In recent years parasitologists have accepted this

interdisciplinary approach of research. A number of papers have

now appeared in which the combined role of drugs and immune

system have been investigated. In the past decade substantial

evidence for the immune dependence of chemotherapy has

accumulated. Immunosuppression is known to reduce the efficacy of

chemotherapy in several parasitic diseases such as rodent malaria

(Lwin et al. , 1987), trypanosomiasis (Frommel, 1988),

onchocerciasis (Bianco et al.,1986) and schistosomiasis (Doenhoff

and Bain,1978). It is also well documented that severe cases of

strongyloidiasis in the immunocompromised individuals often fail

to respond to anthelmintic treatment, and repeated drug treatment

is necessary to obtain a complete cure(Scowden et al.,l978;

Weller et al.,1981; Shelhamer et al. , 1982-, Korgan et al. , 1986)

Although the immune factor involved in influencing the

drug efficacy is not yet determined, it has been reported that

antibodies are synergistically involved in enhancing the efficacy

of drug against schistosomes and malarial parasites. When immune

sera was administered simultaneously with the drug, greater

degree of cure have been achieved in immunosuppresed hosts in

schistosomiasis and malaria inffctiorv (Brindley and Sher, 1987;

Targett, 1985). Cyclophosphamide, the nitrogen mustard derivative

when administered to animals before antigenic stimulus has been

shown to reduce selectively the number of B lymphocytes in

lymphoid tissues while having little effect on the T lymphocytes

(TurJc et al. 1972; Turk, 1973). The B cell response as measured by

antibody production to sheep red cells appears to be markedly

impaired by such pretreatment with cyclophosphamide (Smith and

Hughes, 1974), while T cell function is actually increased (Turk

et al., 1972; Kerkhaert et al., 1974; Lagrange et al. 1974).

Vickerman et al. (1976) observed in T. brucei infection that when

cyclophosphamide was given on 'hird day after infection, a high

non relapsing parasitaemia developed in treated animals. Such

immunological variations have a direct impact on epidemiological

studies as suppression of skin t6st in filarial patients treated

with DEC was observed (Sawada et al.,1968; Katiyar et al., 1974

and Murthy et al.,1978) . Keuffer (1976) observed changes in

haemagglutinating antibodies even after a single dose of DEC. In

Litmosoides carlnii infection when orms are transplanted

intraperitonially the immune status shows variation after DEC

administration (Misra et al.,l982). Effect of DEC on <3MI response

^5

wag also investigated by Piessens et al.(1981), Mistry and

Subrahamanyam (1986). These findings indicate that immune status

of filarial subjects may be altered as a result of DEC

treatment.These studies also indicate that the authenticity of

sero-epidemiological results become doubtful if the host had been

treated with antifilarials previously. Contrary to these

observations Bekhti et al.(1977) reported increased level of

circulating immune complexes in patient's sera after mebendazole

chemotherapy, but specific IgE level decreased. The parasitocidal

activity of mebendazole on the larval stages of E. granulosus

have been observed in experimental animals (Eclcert et al.,1978

and Schantz et al. 1982) . Further, Muller et al,. (1982) and

Schantz et al. (1982) tested the efficacy of this drug in human

cystic and alveolar echinococcosis. In order to assess the effect

of this drug on antibodies level a study was carried out on

patients undergoing prolonged treatment with mebendazole and

observed a decreased level of antibodies as well as total serum

IgE concentrations (Woodtli et al.,1985). In E. granulosus

infection decreased antibody titer was also observed following

chemotherapy (Dessaint and Capron, 1982; Schantz et al.,1982;

Loscher, 1983).

In another study Gottstein et al^ (1984) investigated

parasite specific immunoglobulins by ELISA in a number of

patients of E. granulosus before and after mebendazole treatment.

In majority of cases IgG decreased and levels of IgA and IgE

gave inconsistent results. However, in progressive disease

Condition the reverse tendencies were observed in the level of

• J "*

Igs. These authors are unable to explain the cause of decrease in

Igs under chemotherapeutic pressure. OzeretskovsJcaya et al. (1979)

suggested that such effects may be a consequence of reduced

antigen release or to be a toxic effect of the drug on immune

system.

Nolla Panades et al. (1980) have reported after treating otie

patient with multicystic liver cyst that antibody level declined

to negative values following chemotherapy with mebendazole while

Kovrova et ai. (1979) have suggested that mebendazole in

experimental echinococcosis is responsible for a reduction of

specific humoral immunity and a stimulation of specific cellular

immunity.

Kamath et al. (1985) also observed a decline in antibody

titer in A. ceylanicum infected hamsters following mebendazole

therapy, whereas levamisole treated hamsters could restore the

titre later because of its immunopotentiating action (Janssen,

1976). Similarly Khan et al,. (1991) have also investigated

alterations in immunological response before and after

chemotherapy in hamsters infected with A. ceylanicum. Humoral

response assessed by ELISA depicted gradual depletion in antibody

titre following treatment with mebendazole, albendazole and

pyrantel pamoate.

In addition to this, such combined effects of drugs on the

immune system and their efficacy have also been carried out on

various species of nematodes particularly on Nippostrongylus

brasiliensis in which DEC either inhibits sJcin test or

significantly increase susceptibility of subsequent infection. In

L i

both the cases there must be obvious decline in the antibody

titer ( Katiyar et al. , 1974, 1978, 1985; Chatter jee et al.,1976,-

Murthy et al., 1978). In another filarial worm, D. immitis marked

increase in ELISA titers has been observed subsequent to an

initial thiacetarsamide treatment and the titre gradually

decreases in drug treated dogs (Grieve and Knight, 1985).

Similar changes in immune system have also been reported

from S. mansoni infection by using fluorescent antibody detection

techniques (Ambroise-Thomas et al., 1970). In Trichinella spp.

infection antibody titer show changes following anthelmintic

treatment (Corba et al., 1977). Further it has been observed

that administration of compound 48-80, made the immune mice

susceptible for S. mansoni infection (Ogilvie, 1974 and Dean et

al.,1976).

Such immunosuppressing effects of post treatment period are

generally being c( sidered as major hurdles in the interpretation

of sero epidemiological data of a particular endemic region. In

Fasciola hepatica. stage specific antigens (Tl and T2 bodies)

have also been tested for their antibody response following

diamphenithide treatment (Hanna et a1.,1982). They observed that

the antibody titer against Tl antigen was unaffected by the drug

treatment from 3 to 6 weeks, however, the T2 antibody response

was some what reduced. This shows that types of antigen also play

a significant role in antibody titer. Benex et al. (1973) also

observed a significant reduction in the antibody titer following

anthelmintic treatment in F. hepatica infection in sheep. Some

other workers have also reported that antibody titer drop

z.'

dramatically in rabbits and rats infected with F. hepatica

following rafoxanide treatment (Hillyer and Llano de Diaz,1976

and Hillyer and Santiago de Weil, 1979). Similarly in another

trematode infection, reduction in antibody titer and complement

activity was noticed following rafoxanide or ivermectin treatment

in sheep infected with Hasstilesia (Daugalieva, 1986). Besides

these reports malarial infection and chemotherapy have also been

investigated on these lines. Quinine and primaquine can depress

the lymphocyte response to mitogens such as PHA and to various

antigens (Thong and Ferrante,1978; Gold et al., 1979). Mefloquine

depressed antibody formation but did not affect delayed type

respoBse (Thong et al,. , 1979). In addition to this chloroquine

induces suppression of lymphocytes just like quinine and

primaquine under in vitro condition (Hurvitz and Hirschorn,1965).

It has been reported that chloroquine stabilizes lysosomal

membranes interfering with processing of antigen (Hurvitz and

Hirschorn, 1965 and Lee et al.,1982). Further, the concentration

of chloroquine 0.8 ug/ml is also an important factor in inducing

some immunotoxic effects as interference with release of

interleujcin I from monocytes and thus inhibits generation of

antibody producing cells (Salmeron and Lipsky, 1983).

In some other protozoan infection like Giardia. Entamoeba

•and Trichomonas metronidazole suppresses the CMI reactions, such

as granuloma formation. The effect can be enhanced when it is

simultaneously administered with antigen (Grove et al.,l977).

Comparatively a new drug Cyclosporin A (CsA), a fungal derivative

was developed for clinical use because of its immunosuppresaive

properties. It reversibly inhibits transformation of stimulated

T cells, suppressing both humoral and cell mediated immunity.

This drug is also reported to have antimalarial activity

(Thommen-Scott, 1981). Besides its immunosuppressive and

antimalarial activity, the anthelmintic properties and

immunomodulatory nature have been comprehensively reviewed by

Chappell and Wastling (1992). Because of its profound

immunomodulatory nature andantischistosomal action of CsA is

mediated via an augmented immune response although the usual

effecs of CsA on the immune system are down regulation of T cell

related events (Thomson et al., 1986). CsA can potentiate the

immune response to non parasite antigens under certain conditions

(Aldridge and Thomson, 1986; Webster and Thomson, 1987) and

augmented pulmonary granuloma formation around the schistosome

egg (Metzger and Peterson, 1988). The small number of published

reports on the action of CsA on cestodes reveal that the drug is

antiparasitic against some species while it may be

immunosuppressive for other infecti ns.

Having established that the effectiveness of many drugs can

be increased by the immune response it is logical to try

combining drug treatment with compounds that enhance immunity.

Among different anthelmintics only levamisole was reported to

have immunostimulating action (Merluzzi et al. ,1976; Renoux and

Renoux, 1977). It has been used for chemotherapy of helminths

either alone or in combination with other drugs.

It is evident from the foregoing review of the literature

that, the mode of action of various anthelmintics have been

studied by a number of workers but no generalization can be made

on the basis of present status of our knowledge, because it is

often stated that a particular drug may have multiple modes of

actions. Also the same anthelmintic may behave differently in the

taxonomicaily different group of parasites, thus pointing the

necessity of studying each member separately to investigate the

mode^ of action of various anthelmintics and the possibility of

their broad spectrum efficacy.During the course of this study it

was proposed to investigate the effect of mebendazole,

oxyclozanide and rafoxanide on the membrane bound marker enzymes

of G. explanatum and G^ crumenifer inhabiting two different

habitats. Furthermore, effects of these drugs on the glucose

uptake was also monitored. Oxyclozanide is a known flukicide and

is frequently used, hence, it was decided to investigate its

effect on immune system of laboratory animals.

31

MATERIALS AND METHODS

PARASITE OOLLECTTION: Active and adult Gastrothylax crumenifer and

Giqantocotyle explanatum were collected from fumen and liver

respectivly from Indian water buffalo Bubalua bubalis slaughtered

at local abbatoir. The worms were bVought to laboratory

expeditiously and washed in three changes of Hank's balanced salt

solution (HBSS) without glucose premaintained at 37+2°C.The worms

were immediatly used for isolation of surface plasma membrane and

for other studies.

ISOLATION OF SURFACE PLASMA MEMBRANE: The method used for

isolation of surface plasma membrane was essentially the same as

described by Rahman et al.(1981a) with some modification

suggested by Khan (1991).The membrane disrupting solution (MDS)

constitutes 1* saponin in HBSS pH 7.8.The MDS solution was always

prepared fresh, cetrifuged for 16,000xg for 60 min and the

supernatant was filtered and stored at S C until used.

Fourty worms of each species were rinsed quickly in HBSS,

premaintained at 37+2 "C and transferred to 100 ml flask

containing 20 ml MDS at 4*0. The flask were kept for continuous

shaking on electrically driven shaker maintained at 4 "c.The

surface plasma membranes were removed by continuous shaking (90

cycles per min) for 20 min. After shaking, the MDS containing

worms were tansferred in a 25 ml stoppered tubes and vortexed on

cycloraixer at high speed for 30 seconds.The carcasses of the

parasites were removed and the contents were pas&ea through

'6

Worms + MDS C 1% Saponin ) .

Shaken at 4 C, 20 min. (90 Cycles/ min.)

Vortex (30Sec.)

Carcass Discarded

Pellet Discarded

MDS Surface Plasma Membrane

Centrifuged at 1500j5.g,15 min.

Supernatant i

Centrifuged at 16,000 X g. 30 min.

Pellet S u p e r n a t a n t "^resuspended in Tris HCl.pH 7.4 Discarded I

Centrifuged at 16,000 X g, Ih

Supernatant Discarded

Pellet

TEM Enzyme quantitation F i g . 1: Flow diagram for the isolation of surface plasma membrane .

3 3

coarse sieve.

The filtrate containing maximum foam and plasma membrane

fragments were kept at 4*0 until used for centrifugation.

CENTRIf^GATION: The MDS containing surface plasma membrane were

first c ntrifuged at low speed (1500 x g) for 15 min in a

refrigerated centrifuge (I E C , 25) at 4 ''c.The pellet which

contained mainly debris was discarded and the supernatant was

further centrifuged at high speed at 16,000 x g for 30 min in

BecJcman L8-55 Ultra Centrifuge. The resultant pellet was

resuspended in 0.1 M Tris- HCl buffer (pH 7.0 at 4 "o and

centrifuged again at 16,000 x g for JO min, the buffer was

discarded. In order to wash the pellet and remove the traces of

detergent, the pellet was resuspended in Tris- HCl buffer and

centrifuged for 1 hr at 16,000 x g. After washing, the final

pellet was dissolved in a known volume of 0.1 M Tris - HCl buffer

(pH 7.4) and sonicated for 30 sec under chilled conditions (4 °C)

by using a 5 mm probe of ultrasonic disintegrator ( Ralsonic,

India).

TRANSMISSION ELECTRON MICROSCOPY: The membrane pellets, recovered

after the treatment of detergent for 20 min, were fixed for 3 h

at 4 °C in 4X (v/v) glutaraldehyde in 0.1 M cacodylate buffer

containing 3* sucrose (w/v) and 0.015* CaCljCw/v). After the

primary fixation the pellets were washed three times in buffer

containing sucrose and CaCl^ . Thereafter,the pellets were post

fixed in 1 % (w/v) osmium tetroxide for l h, dehydrated in graded

sereis of acetone to propylene oxide and embedded in spurr resin.

Ultrathin sections were cut on LKB -IV ultramicrotome.

• I r

3';

collected on bare copper grids and double stained with uranyl

acetate and Reynolds lead citrate sections were examined using

JEM 100 CX electron microscope at various magnifications.

PROTEIN ESTIMATION: The total protein content in isolated surface

plasma membrane fraction was determined by dye binding method c

Bradford (1976) as modified by Spector (1978) using bovine serum ft-

albumin (BSA) as standard.

PREPARATION OF HOMOGENATE: Adult worms were collected , washed in

two changes of HBSS, blotted and weighed. Ice cold 0.25 M sucrose

was added to give 10* (w/v) extract and then homogenized using

Potter-Elvehjm tissue homogenizer with teflon pestle kept in ice

bath. These homogenates were centrifuged at 1500 xg for 20 min at

4''c. The supernatant was stored at 4°C until required for enzyme

assay.

ENZYME ASSAY:

ACID PHOSPHATASE (EC 3.1.3.2): Acid phosphatase was assayed by

the method of Bergmeyer et al.(1974) using p-nitrophenyl

phosphate as substrate. The total reaction volume of 1.1 ml

comprised of 0.05 M acetate buffer pH 5 containing 8 mH p-

nitrophenyl phosphate (pnp), 0.05 M Tris- HCl pH 7.0 and protein

sample.The reaction mixture was incubated for 30 minutes at 37*'c.

Thereafter reaction was stopped by the addition of 8.9 ml of 0.02

N NaOH. The liberated p- nitrophenol was measured at 420 nm.

ALKALINE PHOSPHATASE (EC 3.1.3.1): Enzyme activity was determined

by the method as described by Bergmeyer (1974) using same

substrate as in acid phosphatase. Total volume of reaction

mixture was l.2 ml comprising o. 1 ml, 0.05 M. glycine-NaOH buffer

3;j

pH 9.5 containing 5 mM substrate (pnp).

Protein sample and total volume was adjusted by Tris - HCl

buffer pH 7.0. Reaction mixture was incubated for 30 minutes at

37 'C and thereafter the reaction was stopped by the addition of

8.8 ml of 0.02 N NaOH. The liberated p-nitrophenol- was measured

at 410 nm.

Enzyme activity of acid and alkaline phosphatases was

determined by previously prepared standard calibration curve of

known concentration of p-nitrophenol. Activity of acid and

alkaline phosphatases was expressed as AJ gm p-nitrophenol

liberated / mg protein /min.

Y-GLUTAMYL TRANSPEPTIDASE (EC 2.3.3.22): The enzyme activity has

been determined according to the method of Szasz (1974).

The assay mixture (total volume 2.1 ml) contained 4.2 mM L-Y

glutarayl-p-nitroanilide, 52.5 mM glycylglycine, 10.5 M MgCl, 50

mM ammediol buffer pH 9.3 and protein sample (volume adjusted to

0.1 ml with ammediol buffer). Simultaneously, blank devoid of

protein sample was also prepared. The change in absorption

indicating the liberation of p-nitroaniline recorded as 405 nm on

Spectronic -1001.

The specific enzyme activity was expressed as n mole p-

nitroaniline liberated / mg protein / min.

AMWOSINE TRIPHOSPHATASE (Mg dependent) (EC 3.6.1.3): Enzyme

activity was determined as estimating release of inorganic

phosphorus in reaction mixture by the method of Ryre (1975) as

modified by Rahman et al.,(1981 b); Verna and Frati (1983). The

reaction mixture (total volume 2.2 ml) comprising of 0.05 ml

protein sample , l ml of 92 mM Tris-HCl buffer pH 8.5, 0.5 ml of

5 mM MgClj. Total volume of the mixture was adjusted by Tris-HCl

buffer pH 7.0. After 10 minutes of equilibration of reaction

mixture at ST'C in shaking water bath the reaction was started

by the addition of 4 mM ATP (Disodium salt, Sigma Chemical

company U.S.A.). After 15 min of incubation at ST^C, the reaction

was stopped by the addition of ice cooled 10» TCA.Then the tubes

were centrifuged at 5000 x g and supernatant was assayed for Pi.

PHOSPHORUS ESTIMATKW: Phosphorus was estimated by the method of

Rouser et al.(1970).The samples were prepared as follows: 0.1 ml

of cone. sulphuric acid was added to 0.2 ml of supernatant /

standard solution and heated in boiling water bath for 2 min

then cooled at room temperature and 0.05 ml of 70% perchloric

acid was added and tubes were again boiled for 2 min. After

cooling, 2 ml of DDW followed by 1 ml of 1* ammonium molybdate

and 1 ml of 1% of ascorbic acid (freshly prepared) were added.

The contents were mixed by vortexing and heating at room

temperature at 70°C for 10 min. Finally after cooling the tubes

at room temperature, absorbance was read at 820 nm. Quantity of

phosphorus was determined by previously prepared standard curve,

using KH^PO^in range of 1-10 /U gm.

ANTHSLMINTICS TESTED:

1. MEBENDAZOLE ( Methyl-5-Ben2oyl-Benzimidazole-2-carbamate) was

dissolved in dimethyl sulphoxide (DMSO) because it is a safe drug

solvent for benzimidazo i.e compounds for in vitro studies (Ahmad

and Nizami, 1983). Final concentration of DMSO in each incubation

aixture was 6* . Controls were also run simultaneously in each

3 7

study containing equal volume of DMSO.

2. OXYCLOZANIDE (2,2'-Dihydroxy-3,3'5.5'6-Pentachlorobenzanilide;

supplied by Imperial Chemical Industries Limited, Great Britain)

is an ethanol soluble compound, each concentration of this drug

was dissolved in ethanol. Controls were also run simultaneously

in each experiment. The final concentration of the ethanol was

approximately 3 \ in each incubation mixture.

3. RAFOXANIDE ( Supplied by Merck, Sharpe and Dohme Research Lab,

U.S.A ) was also dissolved in ethanol. The final concentration of

ethanol was approximately 3 * in each incubation mixture.

Controls were also run simultaneously with equal volume of

ethanol in each experiment.

To study the effect of drugs on membrane bound marker

enzymes of isolated surface plasma membranes of G. crumenlfer and

Q- explanatum each compound was added to the incubation mixture

directly to maintain the drug concentration in terms of nano

moles viz:

Mebendazole 202 nmoles in 150 /ul of DMSO

Oxycolzanide 112 nmoles in 70 AJI of ethanol

Rfoxanide 122 nmoles in 150 AJI of ethanol

Enzyme activity was then measured keeping the total assay

volume constant in case of each enzymes mentioned earlier. The

enzyme activities of the total parasite homogenate were estimated

in order to compare with enzyme levels of the membranes. The

statistical significance of the data was analysed by Student's t-

test (Sokal and Rohlf, 1981).

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^H- GLUCOSE UPTAKE BY LIQUID SCINTILLATION COUNTING:

Tnis study was carried out in rumen amphistomes, G.

crumenifer. Freshly collected worms were thoroughly washed in

HBSS and preincubated for 3 h without glucose. In order to study

the effect of different drugs ( mebendazole, oxyclozanide and

rafoxanide) on glucose uptake, three different concentrations of

these drugs were used in HBSS and the final radioactive glucose

concentration of l Ai Ci/ml (sp. activity 24 Ci/m mole) was

maintained in the incubation medium.

Four worms of same size were incubated in medium for 10 min

at 37-t-2*'c. Thereafter the incubated worms were immediately rinsed

and vortexed three times in ice cold saline without any labelled

material in order to remove adhered isotope. Thereafter the worms

were soaked on absorbent paper and kept for 24 hours in absolute

alcohol for extraction of incorporated material. To each

scintillation vial 8 ml scintillation cocktail containing 4 g PPG

(2,5-diphenyloxazole) and 50 mg POPOP (1,4-bis(phenyl-2-oxazolyl)

benzene,2,2-phenylene bis (5 phenyloxazole) per liter of toluene

was added and counted for 1 minute in Wallac, 1410 Liquid

Scintillation Counter, Sweden.

REVERSED ROCKET IMMUNOELECTROPHORESIS (RRIE): In order to monitor

the effect of oxyclozanide on the immune response the antibodies

were raised against the parasite. For this study the soluble

fraction of the total homogenate of the parasite was initially

usea to develop and to collect the hyperimmune sera of the

rabbit. Simultaneously the same rabbit was also given known

amount of drug. Tht concentration and other protocol details are

t.)

given below. The quantification of the level of precipitating

antibodies against plasma membranes was carried out by using

reversed rocket Immunoelectrophoresis. This tecnique is

essentially the same as described by Laurell (1972) as modified

by Hoyeraal et al.(1975). In this method the antigen was mixed

with molten agarose gel and antisera were placed in the wells at

one edge of the gel. After electrophoresis the precipitation

bands in the form of rockets were analysed.

CiOLLBCrriON OF HYPER IMMUNE SERA: In order to develop hyper

immune sera against the soluble fraction of G. crumenifer,

freshly collected parasites were homogenized in 0.1 M phosphate

buffered saline, pH-7.4, centrifuged at 5000 x g and the

resultant supernatant was sonicated for 30 sec at 4 "C. Protein

was estimated by the method of Spector (1978) as described

earlier. The homogenate was emulsified with equal volume of

freund's complete adjuvant (Difco) and injected subcutaneously at

four different sites along the aide of spinal cord. Firrt

inoculation consists of 4 mg protein. Second and third boosters

were given at an interval of 25 days having protein concentration

2.5 mg/ml. Finally, hyper immune sera were collected after 10

days of third booster. For preimmunised sera, blood was collected

from ear marginal veins of the rabbit before inoculation of

parasite the antigen.

To determine the effect of drug on the humoral response,

oxyclosanide was orally administered (7mg/kg body wt) in two

experimental rabbits. First dose was given four days before any

antigen inoculation, following five consecutive doses at a

"i I

regular interval of 10 days. Control sera used was without any

drug treatment.

PREPARATION OF ANTIGEUS: For the preparation of antigen surface

plasma membranes were isolated as mentioned earlier. The surface

plasma membrane pellet was used t;o test the antibodies raised

against the total homogenate of the same parasite. In order to

prepare antigen partially purified surface plasma membranes of G.

crumenifer were disrupted and sonicated in known volume of tris-

HCl buffer, pH-7,4.

PREPARATION OF GEL: For preparing an agarose gel, 0.9* agarose

(BDH-Electran) was molten in 0.05 M citric acid phosphate buffer,

pH-5.2 containing 2 mM calcium lactate. The antigen was diluted

by adding citric acid phosphate buffer and mixed with molten

agarose.The concentration of the antigen in gel was kept at 0.33

mg/100 ml gel. Thoroughly mixed antigen in molten agarose was

poured on a glass plate mfisuring 2x8x15 cm and having 3 mm

thickness of the gel. Thereafter the plate was kept at 4 "c for

30 min. A row of wells measuring 5 mm diameter was punched having

1 cm distance between each well on one edge of the gel plate. In

first five wells control serum (serum without drug treatment) was

applied with a concentration of 10 Ail, 20 /ul, 30 Ail, 40 AI1 and 50

Ail respectively without any further dilution. The next five wells

were loaded with drug treated undil :ed serum with the same

concentration as mentioned above. After loading the control and

test sera, electrophoresis was carried out at a ostant supply of

4 m amp current per well for 6 h without any cooling device. In

all experiments, gel without antigen was used as negative

control. In order to form a bridge between cathode and anode

filter papers were aoalced in running buffer (0.05 M Citric acid

-Phosphate buffer) and placed on the immunoelectrophoreis

assembly so that the antisera containing well remain towards

anode.

STAINING OF GEL: After electrophoresis the gels were removed and

immersed in 5% sodium citrate solution for 10 min so that, false

precipitation may dissolve. Slides containing the gel were then

washed three times by immersing in physiological saline at room

temperature for at least 12 h. followed by immersion in distilled

water for 10 min to remove the adhering salts on the gel if any.

The slides were then wrapped inpreviously moistened filter papers

(Whatman No. 1) with distilled water and kept in thermostat

premaintained at 37 °C for drying. The dried gels thereafter

immersed in amidoblack staining solution ( consisting of 0.1 mg

amidoblack in 20 ml glacial acetic acid and made upto 1 litre

with distilled water). The slides remained dipped for 20 to 30

min until well stained arcs in the form of rocket appeaered. For

further refinement of the results, slides were rinsed with

distilled water and dipped in a clearing solution ( consisting of

ethyl alcohol - 400 ml, glacial acetic acid - 100 ml and double

distilled water - 1000 ml) for 30 - 40 min until a clear

background appears. The plates were photographed with a Canon AE-

1 camera using ORWO, 125 ASA, black and white film.

RESULTS

ISOLATION AND PURITY OF THE MEMBRANES

The membrane pellets recovered after 2 0 min of treatment of

worms with saponin were subjected for transmission electron

microscopy in order to check the purity and degree of

contamination. It is evident from the electron micrographs of the

isolated membranes that membranes of both the amphistomes possess

basic structural organization which is similar to that reported

from other typical plasma membranes. They are basically

trilaminate, showing a bimolecular electron dense lipid layer in

between with associated translucent protein layer (Plate 3

and 4). At some places the Isoated membranes become puffed or

expanded, which may be due to hyposmatic effect of MDS indicating

that these membranes follow chemiosmotic hypothesis which has

been reported for other cellular membranes. No cytoplasmic

junctions or tight junctions were observed. At places dense

coagulated bodies have been noticed which give an appearance like

desmosomes but such bodies may also be due to the effect of

detergent. At higher magnification holes or pits like appearance

were noticed which could be due to the differential effects of

detergent on different lipid moities.

Thus it can be concluded from the present results that

saponin is a suitable detergent for the isolation of surface

plasma membrane of amphistomes and 20 rain treatment is optimum in

order to obtain appreciable quantity of membranes-with purity and

least contamination. In addition to this the vortexing time and

Plate 3: Transmission electron micrographs of surface plasma membranes isolated after 20 min of saponin treatment: GastrothYlax crumeniier : a: showing membrane yield at low magnification (scale bar: 0.5 um). b: showing membrane structure at high magnification (scale bar : 0.2 um). Gigantqcotyle expJLanaturn -. c: showing membrane yield at low magnification, d: showing membrane stucture at high magnification, small arrow: trilaminate membranes, arrow heads: desmosome like appearance and circles: puffed membranes.

44

Q ^>f - > . , W

'^y'Jir

"^••rlvf'

(5)2. J •«• »««^ {'

.-•ii^J / 3 ••> ^ ^ .

r-v:

4

A ^ • \

Wft

f

^

• -r

43

Transmission electron micrograph of surface plasma membranes of Gastrothylax crumemfer after saponin treatment showing trilaminate structure ( arrow ), puffed membranes (circle),and pores (arraw Heads ).

PLATE- 4

the centrifugation protocol adopted in this study was found to be

appropriate for optimum yield. However the transmission electron

microscopy alone does not provide any conclusive evidence of

membrane yield.

Therefore, various memi me bound enzymes have also been

used as parameter for the characterization of membranes. On

comparing the enrichment of membrane bound marker enzymes with

the enzyme activity of the soluble fraction of the worm

homogenate, it is evident from the present study that the level

of enzymes of membranes showed many fold increase than the total

homogenate. The degree of enrichment varies from 3 to 12 old

(Table. 1 and 2). The order of maximum enrichment of various

enzymes also show differences i,e acid phosphatase > alJcaline

phosphatase > Y-GTP > ATPase in G. explanatum. While in G.

crumenifer the sequence is different i.e. acid phosphatase >

ATPase > Y-GTP > alkaline phosphatase. It can be concluded from

these results that many fold enrichment of the known membrane

bound marker enzymes indicate that the specified isolation and

purification procedures are optimal for the yield of membrane

fraction.

QUANTIFICATION OF MEMBRANE AND SOLUBLE ENZYMES:

The quantitation of the primary marker enzymes of the

surface plasma membranes isolated after the treatment of the non

ionic detergent (Saponin) are summarized in Table 1 and 2. The

levels of acid phosphatase is higher than the alkaline

phosphatase in both soluble and membrane fractions of the

amphistomes. However, quantitative differences were observed in

^ 7

Table 1. Enrichment of surface plasma membrane enzyme activities in ^ comparison with fresh worm homogenate ofG. explanatum

Enzymes Homogenate Membrane Fold Enrichment

ACPase

ALK Pase

ATPase°

Y-GTP*

3.700+0,

0.164+0,

0.466+0,

1.660+0,

.23

.02

.006

.39

45.45+3.

1.378+0.

1.540+0.

5.960+0,

. 15

, 15

.05

.77

12,

8.

3.

3.

.28

.40

.30

.59

* Values in ugm pnp liberate /mg protein/ min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmole p-nitroaniline released/mg protein/min + SD

43

Table 2. Enrichment of surface plasma membrane enzyme activities in comparison with fresh worm homogenate of G. crumenifer.

Enzymes Homogenate Membrane Fold Enrichment

ACPase 1.69+0.04 20.46+1.42 112.10

ALKPase 0.37+0.02 0.67+0.038 1.81

ATPase 0.24+0.01 0.95+0.07 3.95

Y-GTP 1.20+0.23 4.26+1.43 3.55

* Values in ugm pnp liberated/mg protein/min jt SD o Values in mg Pi released/mg protein/min + SD • Values in nmole p-nitroaniline released/mg protein/min + SD

43

the level of the various enzymes in the two fractions of G.

explanatum and G. crumenifer.

In the soluble fraction among the various enzymes assayed,

acid phosphatase, Y-GTP and ATPase levels were found higher in G.

explanatum than G. crumenifer. While alkaline phosphatase

activity was greater in rumen amphistome than the liver

amphistome.

Whereas in the membrane fraction acid and alkaline

phosphatases, Y-GTP and ATPase levels were found higher in G.

explanatum than G. crumenifer. On comparing the enzyme data of

membrane and soluble fractions of both the parasites under study,

it is evident that with the exception of soluble alkaline

phosphatase all other enzyme levels were found higher in G.

explanatum.

EFFECT OF ANTHELMINTICS ON ENZYMES:

Some known anthelmintics belonging to benzimidazole and

salicylanilide group whose biochemical mode of actions are

known, were tested on the membranes of the araphistomes because

surface plasma membranes are being considered as primary target

of drug action.

MEBENDAZOEiE: Mebendazole is a known anthelmintic whose mode of

action is attributed with the blocking of transtegumental uptake

of glucose and binding with the tubulin of microtubular system.

It has been observed that in soluble fractions mebendazole has no

effect on the enzymes understudy in G. crumenifer (Table- 3)

while in G. explanatum with the exception of ATPase all other

•enzymes are significantly stimulated (Table- 5).

Table 3. Effect of athelmintics on membrane bound marker enzymes of G. crumenifer ( soluble fraction)

bO

Drugs Cone, (nmoles)

ACPase ALKPase ATPase Y-GTP

NORMAL

MBZ

OXCL

RFZ

0.0 1.69+0.04 0.37+0.07 0.24+0.01 1.20+0.23

202 1.71+0.15 0.39+0.07 0.23+0.01 2.91+1.49 (p>0.05) (p>0.05) (p>0.05) (p>0.05)

112 1.96+0.04 0.40+0.015 0.23+0.002 0.95+0.36 (p<0.001) (p>0.05) (p>0.05) (p<0.001)

122 2.14+0.09 0.40+0.015 0.26+0.028 3.99+0.75 • (p<0.001) (p>0.05) (p>0.05) (p<0.01)

» Values in ugm pnp liberated /mg protein/min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmoles p-nitroaniline liberated/mg protein/min + SD p value upto <0.05 is considered as significant.

Table , 4. Effect of anthelmintics on membrane bound marker enzymes of G. crumcnifer ( membarane fraction)

Oi

Drugs

NORMAL

MBZ

OXCL

RFZ

Cone, (nmoles)

0.0

202

112

122

ACPase

20.46+1.42

22.13+1.09 (p>0.05)

21.64+1.84 (p>0.05)

22.28+1.48 (p<0.05)

ALK Pase ATPase Y-GTP

0.67+0.04 0.95+0.070 4.26+1.43

1.40+0.09 0.58+0.030 3.04+1.95 (p<0.001) (p<0.001) (p>0.05)

0.90+0.19 0.93+0.025 3.64+2.52 (p<0.05) (p>0.05) (p>0.05)

0.76+0.057 0.96+0.045 6.28+0.96 (p<0.05) (p>0.05) (p>0.05)

» Values in ugm pnp liberated/mg protein/min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmoles p-nitroaniline/mg protein/min + SD p value upto <0.05 is considered as significant.

Table 5. Effect of anthelmintics on membrane bound marker enzymes of G. explanatum ( soluble fraction).

Drugs Cone, (nmoles)

ACPase ALK Pase ATPase Y-GTP

NORMAL

MBZ

oxca.

0.0

202

112

3.70+0.231 0.164+0.020 0.466+0.006

4.120+0.304 0.270+0.016 (p<0.05) (p<0.001)

4.370+0.179 0.160+0.012 (P<0.01) (P>0.05)

RTC 122 4.450+0.302 0.174+0.014 (p<0.02) (p<0.001)

0.475+0.013 {p>0.05)

0.462+0.033 (P>0.05)

0.474+0.016 (p>0.05)

1.660+0.390

3.720+1.700 (p<0.05)

2.240+0.253 (P<0.05)

43.970+6.17 (p<0.001)

* Values in ugm pnp liberated/mg protein/min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmole p-nitroaniline released/mg protein/min + SD p value upto <0.05 is considered as significant.

Table 6. Effect of anthelmintics on membrane bound marker enzymes of G. explanaturn ( membrane fraction).

Drugs

NORMAL

MBZ

OXCL

RFZ

Cone, (nmoles)

0.0

202

112

122

ACPase

45.45+3.15

46.24+4.87 (p>0.05)

47.22+6.32 (p>0.05)

49.56+6,19 (p>0.05)

*

ALK Pase

1.38+0.15

3.58+0.93 (p<0.01)

1.33+0.13 (p>0.05)

1.43+0.14 (p>0.05)

0 ATPase

1.54+0.05

1.44+0.03 (p<0.05)

1.59+0.06 (p>0.05)

1.66+0.15 (p>0.05)

• Y-GTP

5.96+0.77

9.96+3.08 (p<0.05)

9.99+3.66 (p<0.05)

18.88+7.98 (p<0.01)

« Values in ugm pnp liberated/mg protein/min + SD o Values in mg Pi released/mg protein/min + SD • Values in nmole p-nitroaniline released/mg protein/min + SD p value upto <0.05 is considered as significant.

b-t

In the membrane traction of rumen parasites mebendazole has

no effect on acid phosphatase and Y- GTP. Contrary to this,

highly significant stimulation was noticed in alkaline

phosphatase (Table- 4) where as ATPase of membrane origin

was inhibited in both rumen and liver parasites. I' G. explanatum

membrane bound alkaline phosphatase and Y- GTP are stimulated a

while on acid phosphatase no effect was observed (Table- 6).

These results reveal that some quantitative as well as

qualitative differences are noticed in the two fractions of both

the amphistomes inhabiting two different microenvironment.

OXYCniiOZANIDE: Oxyclozanide belongs to salicylanilide group which

is the most potent anthelmintic used for the control of immature

paramphistomiasis. The mode of action is to block MDH and

uncoupling of oxidative phosphorylation. In the present study,

oxyclozanide stimulates the acid phosphatase of the soluble

fraction, while had no effect on the membrane bound acid

phosphatase of both the parasites (Table 3, 4, 5 and 6).

Alkaline phosphatase of membrane origin of G. crumenifer is

stimulated while in both the soluble fraction as well as in the

membranes of G. explanatum this-drug is unable to induce any

effect (Tables 5 & 6).

The effect of this drug on Y- GTP is quite interesting in

liver parasite. The enzyme of both the fractions is stimulated

(Tables 5 & 6) while in the rumen amphistome Y- GTP of membrane

origin did not show any effect whereas Y- GTP of soluble fraction

was significantly inhibited (Tables 3 & 4).

Oxyclozanide had no effect on ATPase of both the parasite.

J.)

in both the fractions. It is interesting that the differences

observed in the two fractions of both the parasites show some

degree of similarity and dissimilarity reflecting the variation

in the physicochemical properties of enzyme molecules.

RAPOXANIDE: Rafoxanide also belongs to salicylanilide group

which also interferes with energy generation mechanism of the

parasite and has greater affinity of binding with the plasma

protein in both the parasites with the exception of membrane

bound acid phosphatase of G. explanatum. This drug had no effect

on alkaline phosphatase of soluble fraction while producing

stimulatory effect on membrane bound alkaline phosphatase of G.

crumenifer and vice versa in G. explanatum (Tables 3 & 4, 5 & 6).

The Y- GTP is stimulated in both the fractions of parasites

understudy with the exception of membrane bound Y - GTP of G.

crumenifer. Rafoxanide did not produce any effect on ATPase like

oxyclozanide. This indicates that since these drugs belong to the

same group, have some degree of identity in their mode of action.

Besides this, some differences were also observed which can be

explained on the basis of physicochemical nature of the habitat

leading to differences in the membrane organization of the two

parasites.

EFFECT OF DRUGS ON^H - GLUCOSE UPTAKE: In order• to know the

possible role of the membrane bound enzymes in the

transtegumental uptake, the^H-glucose was used in the incubation

medi > in presence and absense of drugs. Different concentrations

of the drugs were used and the uptake was monitored by

scintillation count-lng.

bG

3 Table 7. Effect of some anthelmintics on in vitro uptalce of H

glucose in G. crumenifer.

Drug

CONTROL

MBZ

OXCl

RFZ

Cone, (nmoles)

0.0

202 101 47.2

110 56 24

122 61 28.5

Glucose uptake (u moles/gm dry wt./lO min)

281.87

120.41 184.68 193.73

267.63 366.30 323.02

349.83 298.64 355.86

Values are the mean of 3 replicates

Fig. 3: Effect of mebendazole on in vitro uptake of^H glucose by G. crumenifer. C: control

5/

.a

•a

a \

O a

I

3

OR o u

300 -

260

200 -

160 -

100 -

6 0 -

C 47.2 101 202 Mebendazole concentration (nmoles).

3 Fig. 4. Effect of oxyclozanide on in yit.ro uptake of H glucose

by G. crumenifer. C: control

b8

400

.3 a ^ 300

a

o

a

IS V o

I—I O

200

100

C 110 56 24 Oxyclozanide concentration (nmoles)

Fig. 5, Effect of rafoxanide on in vitro uptake of^H glucose by 5• crumenifer. C: control

b.)

400

0 a o

rs

a

« o o 0 o

300

I 200 a

I M 4^

100

C 29 61 122 Rafoxanide concentration (nmoles).

bO

The results of glucose uptake by G. crumenifer in presence

of different drugs are summarized in (Table. 7). After 3 h of

incubation in HBSS medium, G. crumenifer utilized different

amount of^H-glucose.

Mebendazole produced inhibition in glucose uptake. However,

the degree of inhibition depends on the drug concentration. It is

evident that the inhibition of glucose uptake is directly

proportional to drug concentration (Fig. 3).

It can be seen from the Fig. 4 that the lowest

concentration of oxyclozanide produced significant stimulation of

glucose uptake, and this stimulatory effect increases upto

second higher concentration. But highest concentration showed

significant inhibition of glucose uptake by G. crumenifer as

compared to control.

Rafoxanide also has a stimulatory effect on glucose uptake

t>y G. crumenifer but here a regular pattern was not observed. At

lowest concentration the glucose uptake was found to be maximum

and at next higher concentration it decreases. At highest

concentration glucose uptake again increases significantly. It is

evident from these results that all three concentrations of this

drug produced significant stimulatory effect by G. crumenifer in

vitro (Fig. 5).

It can be concluded from these results that benzimidazole

and salicylanilides show differences in their moete of action as

also observed in the inhibition and stiumulation of membrane

bound enzymes.

b l

CONTROL ^ « • «

EXPERIMETAL M

Reverse Rocket Immunoelectrophoresis of Control and Experimental Sera Against Membrane Antigens of Gastrothylax crumenifer

PLATE 5

Fig. 5. Diagrammatic representation of precipitation peaJcs following reversed rocket immuno-eletrophoresis.

b ')

eOOOSee©^^ xiH 10 20 30 40 50 ' 10 20 30 AO 50

CONTROL EXPERIMENTAL

.)

Table 8. Effect of Oxyclozanide on the antibody titer in rabbit.

Volume

10 ml

20 ml

30 mi

40 ml

50 ml

Height

Control

7.0 mm

10.0 mm

13.5 mm

14.5 mm

15.5 mm

of Peaks (mm)

Experimental

4.5

5.5

6.5

7.5

9.0

Specific Activity (Height/vol. of antiserum)

Control

0.70

0.50

0.45

0.36

0.31

Experimental Ii

0.45

0.28

0.22

0.195

0.18

-— *

ihibition

43

EFFEX3T OF OXYCLOZANIM: QH CIRCULATING ANTIBODIES LEVEL BY REVERSE

ROCKET IMMUNOTLECTROPHORESIS: Finally an attempt has been made

to monitor the antigenicity of membrane protein and to workout

any possible immunomodulating role of oxyclozanide. The

immunomodulatory effect was monitored by asessing circulating

antiboty level through reversed rocket Immunoelectrophoresis.

The results obtained after the reversed rocket

Immunoelectrophoresis are shown in plate 5 and fig. 6. Two

distinct precipitation peaks were noticed when both drug treated

and untreated (control) sera were reacted with membrane antigens.

The ou r precipitation peak was sharp and prominent while the

second was found to be diffuse.This shows that two antigenic

components are present in the membrane antigen detected with

antisera raised against whole worm homogenate in both drug

treated and control animals.

It was observed that height of the rocket peaks were

directly proportional to the antibody concentration present in

the antisera loaded in antigen containing gel (Table. 8). On

comparing the immunoprecipitation pattern a gradual decrease in

the height of peaks corresponding to the antibody concentration

was depicted between drug treated and untreated animals. The

overall suppression in antibodies level were found to be 43% in

drug treated animal sera.

DISCUSSION

It is evident that Saponin was found to be appropriate for

the isolation of surface plasma membranes of amphistomes and

appreciable yield of membranes was observed in 20 minutes of

detergent treatment with least contaminations.

Saponin has been frequently used for the isolation of

surface plasma membranes of Schistosoma sp, (Kusel, 1972;

Cordeiro and Gazinelli, 1979; Podesta & McDiarmid, 1982;

McDiarmid et al, 1983; NOel and Scares De Moura, 1986) and in

some cestodes (C •n et al, 1977; Oaks et al, 1977; Knowles and

Oajcs, 1979; Rahman et al. , 1981 a; Pappas, 1983; McManus and

Barrett, 1985 ). But the concentration and other conditions were

quite variable in the above mentioned studies. However, Saponin

has been reported to be the detergent of choice because at

concentrations below the critical micellar concentration, it has

the ability to enhance the ATPase activity (Stekhoven and

Bouting,1981). Therefore an optimum concentration of saponin

ci.e. 1 *) has been worked out by previous workers which has been

used in various helminths as well as in the present study.

Cain et al. (1977) and Oaks et al. (1977) have used 1%

saponin for 10 min for the iso-lation of membranes with intact

membrane bound enzyme activity. Rahman et al. (1981 a, b) have

also used 1% saponin in hypotonic and hypertonic MDS and obtained

comparatively more pure fractions of the brush border membranes

of H. diminuta with slight mitochondrial contamination. Similarly'

bb

McManus and Barrett (1985) have obtained appreciable yield of

membranes in 1* saponin with 20 min detergent treatment of E

3E3Qyi03yS protoscoleces. All these workers have used 1 x 30 sec

shearing step on a bench top vortex mixer and differential

centrifugation procedures of varying time period. Purity of

surface plasma membranes isolated with saponin may be due to the

fact that saponin has been reported to produce differential

effects on the surface plasma membranes and on the basement

membrane of the tegument. The possible reason of low

contamination in membranes isolated with saponin treatment has

been suggested by Oaks et al.(l977) and Rahman et al. (1981,a).

They have reported that saponin does not appear to affect the

morphological integrity of the basement membranes of helminth

tegument, because a protein containing structure potentially

capable to interact with saponin (Assa et a1., 1973) and then

prevent the release of cellular components in the MDS.

The low contamination in saponin isolated plasma membranes

of amphismtomes may also be due to the ability of saponin to

interact with the hydroxyl roup of cholesterol as also suggested

by Kavanau (1965). Since cholesterol characteristically lowers

the fluidity of hydrocarbon chain in the hydrophobic region and

thus stabilizes membrane structure (Papahadjopoulas etal., 1974).

Therefore it may be possible that amphistomes when treated with

this detergent for short duration, saponin interacts with the

membrane cholesterol thus causing a reordering of endogenous

cholesterol resulting in structural weakening of the surface

b?

plasma memrbranes and relatively mild vortexing can remove the

surface membranes.

Further Kavanau (1965) suggested that interactions between

the hydroxyl group of cholesterol and the unsaturated lactone

rin>:; of the saponin forms complexes, while Lucy and Glauert

(1964) believes that saponin creates pores in membrane and these

pores on fusion forms gaps. Such pores have also been noticed at

some places in isolated membranes of amphistomes in 20 min

saponin treatment by TEM. Appreciable quantity of cholesterol has

also been reported in membranes of amphistomes under study (Khan,

1991) .

The basic structure organization of the surface plasma

membranes of amphistomes understudy is essentially the same as

in the other helminths with the exception of schistosomes (see

review by Lumsden, 1975). The trilaminar structure has been

described by Singer and Nicolson (1972) for other cell memrbanes.

In addition to this the swelling at certain places in the

isolated membranes may be due to the osmotic stress of MDS. These

localized swellings on the isolated membranes suggest that these

portions may actually enclose various types of papillae reported

from the surface of these amphistomes (Dunn et al.,1987) which

may be involved in various physiological action like chemo/or

osmo reception.

Thus it can be concluded from the foregoing discussion that

saponin is the most suitable detergent for the isolation of the

surface plasma membranes of amphistomes with appreciable yield

bS

and least contamination. Further the various membrane bound

enzymes could be used as markers for monitoring the purity of

these membranes.

The qualitative enzymatic differences were noticed in the

membranes of live and rumen amphistomes, which may be a

consequence of the parasitic adaption in physicochemically

different habitats. As McDiarraid et al., (1983) pointed out

that " in th studies of membrane purification it is necessary to

have a marker for identifying the purity of the membrane

fractions. This is usually accomplished by using enzymes unique

to the membrane being isolated." The presence of ^ cious enzymes

in the surface plasma membranes of amphistomes also suggests that

they follow the same enzymatic organization as in S. mansoni

inner bilayer as well as plasma membranes of other cell types.

Our results are in agreement with other reports available on

helminth parasites.

In the present study, acid and alkaline phosphatases, ATPase

and Y- GTP were found significantly high after the treatment with

saponin. It has been suggested that alkaline phosphatase could be

used as a marker for the tegument membranes. In helminths the

presence of alkaline phosphatase are often indicative of membrane

transport mechanism. Roberts et al. (1983) have used the alkaline

phosphatase as a marker for the surface plasma membrane of

Schigtosom. sp Further, Wheater and Wilson (1976) suggestd that

alkaline phosphatase could be used as marker for the surface

plasma membranes. It has been pointed by Simpson et al. (1981)

b3

that 20% of the alkaline phosphatase was located on the surface

of S. mansoni, while Cesari (1974) estimated 75\ of the enzyme

from the surface membrane fraction. Similarly, Roberts et al.

(1983) observed that 34* of the alkaline phosphatase of total

worm homogenate was present in the surface membrane of S.

mansoni. These findings support our results, in which many fold

enrichment of alkaline phosphatase was observed in the isolated

surface plasma membranes, than the fresh worm homogenate.

Acid phosphatase was noticed in appreciable quantity in the

surface membranes of both the amphistoraes under study. Regarding

its function in the membranes Threadgold (1968) sug' 3ted that

the presence of acid phosphatase in plasma membranes are often

associated with transmembrane absorption. Similarly this enzyme

has also been reported from the surface membrane of S. mansoni

and from the basal apical plasma membranes of G. crumenifer and

G. explanatum (Watts et al. 1979; Dunn et a1., 1987). Khan

(1991) have reported the occurrence of ATPase enzyme in isolated

membranes of amphistomes and revealed that this enzyme occurs in + ++ + +

two forms is Na /Mg and Na /K dependent, the former form is + + +

predominant than the latter. Further Na /Mg dependent enzyme is + +

stimulated while Na /K dependent form is inhibited by ouabain. + +

Podesta & McDiarmid (1982) have also observed Mg activated and

ouabain sensitive ATPase in the surface membrane» of S. mansoni.

This shows that these parasites have the ability to make use of

the ATPase system according to their need and physicochemical

conditions of the habitat. At this stage it is difficult to

;• I'j

suggest the nature of transport and occurrence of Na pump, which

require further kinetic studies.

Y-Glutamyl transpeptidase (Y- GTP) is a key enzyme of Y-

glutamyl cycle. It is a membrane bound enzyme responsible for the

transport of amino acids across the membrane. Y- GTP has been

demonstrated in M. benedeni but it was not found in A.

lumbricoides and F. hepatica. Singh et al (1989) reported Y- GTP

in bovine filarial nematode Setaria cervi. The results of Y- GTP

estimation in the fresh homogenate and surface plasma membrane

fractions of parasites under study revealed the higher enzyme

activity in G. explanatum followed by G. crumenifer.These results

are further supported by Abidi (1990) who have also reported the

higher activity of Y-GTP in G. explanatum than G. crumenifer.

The presence of higher concentrations of acid and alkaline

phosphatases, ATPase and Y- GTP in the surface plasma membranes

of liver parasite G. explanatum than the rumen trematode G.

crumenifer, can be explained by taking the habitat into account.

Liver parasite live in nutritionally rich habitat where simple

nutrients such as glucose and amino acid are readily availble, it

appears that absorption and transfer of nutrients are performed

by the tegument.

Quantitative differences were also observed in the

distribution of acid and alkaline phosphatases in membrane as

well as in soluble fractions of the two amphistomes under study.

It was found that acid phosphatase is much active than the

alkaline phosphatase in both the parasites. Most of the studies

on adult trematodea have also indicated that acid phosphatase is

more active than alkaline (Von Brand, 1973; Cesari, 1974; Probert

& Lwin, 1974) .

It is evident from the results that the drugs under study

have variable effects on soluble and membrane bound enzymes of

both the amphistomes. Qualitative as well as quantitative

differences were observed in the soluble and membrane fractions

of two parasites. These differences may be attributed to the

different physicochemical nature of the habitat in which these

trematodes inhabit. Alternatinely the possible reason could be

the differences in properties of soluble and membrane bound

enzymes. Generally the differences are explained on the basis of

change in substrate specificity, optimum pH, Km and general

stability. The soluble enzymes exist in polar hydrophobic

environment of high dielectric constant. A variety of molecules

including substrates, other metabolities and ions can approach to

the soluble enzymes easily. In contrast, the membrane bound

enzymes may be largely embebded in a li.pophilic region of low

dielectric constant, with little opportunity for interaction with

smaller polar molecules. The membrane bound enzymes exist in a

relatively stable environment, the nature of which is determined

by the molecular arrangement of the membranes. Changes in

membrane composition or structure will modify the environment of

the enzymes and thus affect the regulatory system (Harrison &

Lunt, 1980).

Mebendazole has a stimulatory effect on acid, alkaline

iZ

phosphatases and Y - GTP of soluble and membrane frations, except

acid phosphatase of membrane origin of G. explanatum while in G.

crumenifer only alkaline phosphatase was stimulated. These

results can be explained by the fact that benzimidazoles are

lipid soluble proton conductors that affect the artificial and

natural membrane system (McCraclcen & Stillwell, 1991). Therefore

mebendazole may release membrane bound enzymes and expose them to

the available drug action. The differential stimulatory effect of

the mebendazole can also be explained by taking the lipid

composition of the membrane of these parasites into

consideration. Khan (1991) has reported that liver tremato^ G.

explanatum has more lipid content than the rumen parasite G.

crumenifer in their surface plasma membranes.

Enzyme stimulation may also be the consequence of damage

and binding of the drug with tubulin moiety, resulting into

release of membrane bound emzymes. As it is well documanted that

membendazole binds selectively to parasite tubulin and therefore

tubulin is considered as the primary chemotherapeutic target

(Kohler & Bachmann, 1980,1981; Ireland et al., 1982; Coles,

1983). Effects of mebendazole to microtubule assembly can

additionally prevent the translocation of secretory products

within the tegument (Bogitsh, 1977; Bogitsh & Carter, 1977;

Borgers and Verheyen, 1976) as well as events which may be

related to the drug induced morpholigical disruption of tegument

and subtegument. It may also possible that mebendazole induces

the release of the membrane bound enzymes and become accessable

/3

to drug molecules producing appreciable marked effects. Further,

Ahmad et al. (1987) have also reported some topographical changes

due to mebendazole in G. explanatum under in vitro conditions.

Besides these effects, mebendazole has inhibitory effect on 3

membrane bound ATPase of both the parasites and uptake of H-

glucose uptake across the tegument of G. crumenifer was also

inhibited. These results are in agreement with the previous

studies carried out on Ascaris suum and Hymenolepis diminuta

and G. explanatum (Van den Bossche, 1972; Van den Bossche and De

Nollin, 1973; McC!racken & Taylor, 1983; Ahmad & Nizami, 1991).

In cestodes the location of transport system undoubtedly be the

tegument (Pappas and Read, 1975; Pappas, 1983), while in

amphistomes Abidi (1990) and Khan (1991) also suggested that

different loci are involved in the transtegumental uptake by

using pulse chase technique of antoradiography as well as by

transport kinetics of some labelled nutrients . These authors

further suggestded that saturable carrier mediated uptake with

the involvement of diffusion component in amphistomes and follow

a saturation kinetics. Similarly cambendazole and mebendazole

also inhibit the tegumental uptake of glucose by M. expansa as

well as affecting a variety of facets of energy metabolism including reduced ATP synthesis and adenine nucleotide turnover

3 (Rahman & Bryant, 1977) Inhibition of H - glucose uptake in G.

+ ++ crumenifer can be correlated with the inhibition of Na - Mg

ATPase system of membrane origin. This further confirms that a

carrier mediated transport is involved and ATP is required. As

'I'i

McManus and James (1975) have reported active transport of

monosaccharides by larval trematodes of Microphallus similis.

Uglem & Read (1975) have also reported the coupled movement of 2 +

deoxyglucose with Na in S.mansoni. It is further suggested

that glucose uptake by G. crumenifer occurs in part, through a +

Na coupled transport system.

Oxyclozanide is a Jcnown flukicide and unconples the oxidative

phosphorylation by blocking ATP synthesis and inhibiting MDH

activity of trematodes (Corbett & Goose, 1971; Probert et al.,

1981). Besides metabolic effects, oxyclozanide is also known to

exert a variety of neurophysiological disorders. Such effects of

oxyclozanide has also been observed in F.hepatica and G.

explanatum (Fairweather et al., 1984; Ahmad & Nizami, 1990).

Further in vitro effects of oxyclozanide on motility of G.

crumenifer reveal that this compound induces disruption in

muscular contraction and cause spastic paralysis within 3 min

(Ahmad & Nizami, 1992). This shows that this drug takes some t me 3

to produce its effect. However, in the present study of H -

glucose uptake in presence of oxyclozanide by G. crumenifer a

concentation dependent effect was observed. At a given

concentration of 24 and 66 nmoles, there was stimulation in 3

glucose uptake, whereas at 110 nmoles, a marked inhibition of H

glucose uptake was noticed. These results provide indirect

evidence that higher drug concentration causes a spastic

paralysis leading to inhibition of glucose uptake. Further,

Ahmad et al., (1987) have reported that oxyclozanide produced

V5

roost pronounced damage at the surface of G. explanatum.

Oxyclozanide causes deformation and disappearance of surface

papillae particularly on dorsal surface where deep lesion in

which parenchymatous and internal organs were exposed. Peeling

of tegume al surface and inner lining of acetabulum was also

noticed. Deep cater like lesions were observed over the exposed

parenchymatous tissues. The topographical disorders observed may

interfere in the absorption of micromolecular nutrients.

Stimulation of acid and alkaline phosphatases of soluble fraction

of both the parasites may be the consequence of topographical

damage resulting in release of bound enzyi.js, while there was no

effect on ATPase of both the parasites. Therefore surface plasma

membrane bound enzymes seem to be destroyed by the action of

this drug, however reported effects on oxidative phosphorylation

or uncoupling reactions may be the conseqeunce of the exposed

tissue. Therefore it is likely that a single drug may have

variable effects depending on the concentration of drug. 3

Inhibition of H-glucose uptake may be a consequence of

topographical damage or metabolic disturbances due to alterations

in the enzyme molecules.

Rafoxanide has been used as flukicide. It is evident from

the results that rafoxanide has no effect on acid, alkaline and

adenosine triphosphatases of the membrane fraction of G.

explanatum while in G. crumenifer, this drug has a marked

stimulatory effect on acid and alkaline phosphatases of the

membrane fractions having no effect on ATPase. On comparing the

vr,

effect of this drug on soluble fraction enzymes, it was found

that acid phosphatase and Y-GTP of the G. crumenifer are

stimulated and acid, alkaline phosphatases and Y-GTP of G.

explanatum are stimulated. Previous studies on F. hepatica have

shown that this drug effects intermediatry steps of metabolism

and end product formation in. main energy producing pathway. It

was pointed out that rafoxanide diminishes ATP synthesis,

resulting in increased carbon flow, followed by depletion of some

internal intermediates pool of the pathway as further metabolism

of succinate is also inhibited (Cornish & Bryant, 1976 b).

Further, Probert & Lwin (1974) have also studied the effect

of rafoxanide and nitroxynil on phosphomonoesterase of F.

hepatica. The refaxanide at 20mM was reported to have no effect

at acid or alkaline pH. 3

In presence of rafoxanide, H-glucose uptake by G.

crumenifer show a marked stimulation at various concentrations of

the drug. stimulation of acid and alkaline phosphatases of

membrane fraction in G. crumenifer may be indirectly attributed

to the transport of glucose under study, because phosphatases

have frequently been associated with absorptive functions at the

parasite surface. Cornish et al.(1977) have studied the effect of

rafoxanide on the energy metabolism of F. hepatica in vivo and

reported that glycogen and ATP levels diminished in 24 hr

treated group. The significar decrease in glycogen content

suggests that it was utilized to maintain the energy levels of

the flukes, but such effects were not or ^rved under in vitro

7 7

conditions in F. hepatica. (Cornish & Bryant, 1976 b). This shows

that the drug has variable effects under in vitro and in vivo

conditions.

Y - GTP is an another important membrane bound enzyme which

plays a key role in the transmembranosis of amino acids across

the cell membranes. The amino acid transport involving the Y-GTP

is an example of group translocation in which amino acid binds to

the enzyme protein and the complex is transported to the inside

of cell where amino acid is released (Lehninger, 1975).

In the present study all the three drugs affect the Y-GTP

and induce stimulation in of the soluble and membrane fractions

in G. explanatum. whereas in G. crumenifer these drugs either

stimulate or had no effect, with the exception of Y-GTP of

soluble fraction, where it is inhibited significantly.

Review of the literature indicates that Y-GTP is

differentially distributed among helminths. This enzyme has been

reported from M. benedeni and body wall of Setaria ceryi

(Barrett, 1981 and Singh et al., 1989). The results of present

study further indicate that Y-GTP level is higher in liver

amphistome than rumen. Glenner et al., (1962) have reported higher

level of Y-GTP from loop of Henle and epithelial lining of the

intrahepatic bile duct. This enzyme occurs both in the soluble

and membrane bound form. It is therfore G. explanatum has higher

level of this enzyme. In the present study by and large, all the

three drugs stimulate this enzyme in order to produce glutathione

which is a reducing agent and plays a domjaant^ role in

fr :.,., ^^

detoxification mechanism, apart from its dominant role in

transmembranosis.It is also expected that under in vivo

conditions, low reducing condition which are essential for

survival of parasites is provided by this pathway (Smyth and

Halton, 1983). This leads us to conclude that in amphistomes,

detoxification mechanism may be operative, which require further

study.

The foregoing discussion on the effect of various drug on

enzymes of soluble and membrane fractions leads us to conclude

that no generalization is possible, and the mode of action and

the degree of efficacy depends on the molecular and biochemical

configuration of membranes, binding nature of drug molecule and

physicochemical conditions of the microhabitat. Since the members

of salicylanilide group are known to induce their effect when

they are bound with the protein moiety, similarly the

benzimidazole binding with tubulin moiety are well authenticated

in the literature. However, this fact can not be ignored that

surface plasma membranes are the primary target of the drug

action, and the metabolic disorders which have been reported in

the present study as well as in previous studies may be a

consequence of secondary effects.

In order to ascertain these effects it is essential that in

vivo studies must be carried out on these lines.

Inununochemical studies:

The results of the present study clearly reveal that

oxyclozanide has a suppressive effect on the circulating

ViJ

antibodies against membrane antigen of rumen amphistomes G.

crumenj-fer as evident by the reduction in the peaks of the

rockets drug treated animal sera.

Digenean flukes are surrounded by syncytial tegument

bounded by an exposed plasma membrane (Lumsden, 1975; Erasmus,

1977 and McLaren, 1980). The tegument is an anucleate, cytoplasmic

structure which is continuous with deeper nucleated cell bodies.

The detailed structure of the tegument is strongly indicative of

continuous turning over or shedding of the outer layers which

are replaced by material contained within the secretory bodies

arising from beneath.

The significance of the contribution of surface antigen has

been reviewed extensively by Maizels et al.,(1982) and

Butterworth (1984), they have emphasised that surface antigens

play a critical role in the expression of effective immune

responses to helminth parasites in vitro and probably also in

vivo. The active release of antigens from the surface of

helminths in yivo could have a potentially significant impact on

the immunobiology of the helminth.

Evidences are on record that immunity of the host

synergistically modulates chemotherapeutic responses of the

infective organisms (Ambroise-Thomas, 1974; Corba and Spaldonova,

1974; Grove and Warren, 1976; Tyagi & Murthy, 1986). Conversely,

chemotherapy also alters the immunologic response of the host

(Katiyar et al., 1986; Janssen, 1976). Changes in immune status

whether suppressive or stimulatory have direct bearing on

S i]

infection/disease status.

Humoral immune response plays an important role in defense

against helminthic infections. .(Ogilvie et al., 1978). The

parasite specific immune response of the host is however expected

to be altered follwing anthelm ntic treatment. Previous work with

F. hepatica demonstrated significant decrease in antibody level

following successful treatment and has been reported to be a use

ful indicator of therapeutic success (Hillyer and Santiago de

Weil, 1979). Similar observations were made by other workers

(Grieve and Knight, 1985 and Kamath et al., 1985) in filarial

and hook worm infections.

Immunological mode of action of salicylanilide group has

been reported in some trematode parasites by a number of workers

(Hillyer and Llano de Diaz, 1976; Hillyer and Santiago de Weil,

1979 and Daugalieva, 1986). They have reported reduction in

antibody titer and complement fixation following rafoxanide and

ivermactin treatment in experimental and natural host. Our

present results are in agreement with these workers in respect to

reduction in antibody titer.

Observations of the present study show that oxyclzanide has

a suppressive effect on the antibody titer which was calculated

to be 43* suppersslon in drug treated animal. However it is not

clear how this suppression was brought about.

First possible rc asor may be that oxyclozanide possess an

inherent capacity to neutralize the already formed antibodies.

Secondly, it suppresses the antibody producing cells. Salmeron

Hi

and Lipsky (1983) have also reported the inhibition of generation

of antibody producing cells after chloroquine treatment. At

present stage, it is not possible to suggest whether the

immunosuppression by oxyclozanide is specific or nonspecific

type, without further studies on natural amphistome infections.

It is an established fact that the effectiveness of many

drugs can be increased by the immune response of the host

therefore it is logical to try combining drug treatment with

compounds that enhance immunity. Levamisole, a known anthelmintic

is the only drug which has immunostimulating action. It has been

successfully used for chemotheraphy of mature and immature

paramphistomiasis with oxyclozanide (Schielhorn Van Veen and

Bida, 1975; Chhabra et al., 1977; Prasad, 1983) but there is no

evidence that its immunostimulating properties promote the

effectiveness of this drug.

The present work provides clear evidence that successful

therapy with a drug having no immunopotentiating action will

bring down the antibody level. The presently available drug

(oxyclozanide) for paramphistomiasis is inadequate. Therapy with

this drug leads to a state of immunosuppression in host.

Therefore there is need for newer strategy to potentiate the

activity of existing antitrematodal drug possibly by modulating

(stimulating) the host immune system while treating, as

participation of immune mechanism of host is almost obligatory

for ultimate destruction and elimination of causative organism.

Immunopharmacological agents which influence defence

82

system, can regulate the number, functional activity and

interaction of macrophages, T and B lymphocytes. Leucocytes and

natural killer cells as well as their humoral and secretory

components. Induction of augmented immune response of host with

immunostimulating agents was initially used wi"n success in the

treatment of malignant tumour (Mathe et al., 1969) and only

recently this approach has been evaluated for the treatment of

experimental infections. Based on this concept, Adenolfi and

Bonventre (1985) were able to enhance the antileishmanial

activity of glucantine by a synthetic immunopotentiating compound

(CP - 46 - 665 - 1). Besides this, quite a number of condidate

substances are being investigated. BCG has also been studied

extensively, its immunopotentiating effect has been shown against

tumour and against a variety of protozoal infections notably

Leishmania (Smrkovsici & Larson, 1977). Glucan, a polyglucose

derivative of yeast cell wall is also a known immunopotentiating

agent used against experimental Leishmania donovani infection as

an adjuvant combined with formalin killed promastigotes (Holbrook

et al,. , 1981). Muramyl peptides, structural components of

mycobacterial cell walls, are amongst the most interesting

immunoprotentiators (Adam & Lederer, 1984).

Thus, it is essential that the use of drugs for the control

of parasite must be carefully tested in order to explore

immunopharamacological aspects of the drugs, because frequent

medication with these drugs may increase the susceptibility to

subsequent infection.

S 3 SUMMARY

In the present study effect of some known anthelmintics on

the surface plasma membrane bound enzymes of two amphistomes i.e.

Gastrothy' x crumenifer and Gigantocotyle explanatum inhabiting

two different microhabitats have been investigated. The surface

plasma membranes of the parasites were isolated by using non -

ionic detergent, saponin. The enriched membrane pellets of the

parasites were characterized by the assay of some membrane bound

marker enzymes and trasmission electron microscopy.

Transmission electron micrograph? f both the parasite's

membranes show a typical trilarainate structural organization

similar to other plasma membranes.

Quantitative differences in the level of primary marker

enzymes were observed in the membranes of both the parasites as

compared with their respective homogenate and the former shows

many fold enrichment than the latter. The order of maximum

enrichment of various enzymes in G. explanatum is acid

phosphatase > alkaline phosphatase > Y-GTP > ATPase. While in G.

crumenifer the sequence is different i.e. acid phosphatase >

ATPase > Y-GTP and alkaline phosphatase.

On comparing the quantitative differences between G.

crumenifer and G. explanatum, all the enzyme levels were found

higher in G. explanatum than G. crumenifer. These differences may

De attributed to the physicochemical conditions of the micro -

habitat.

Different drugs used in the present study, produced

differential effects on the membrane bound enzymes in both the

parasites, ranging from stimulation or inhibition to no effect.

Mebendazole, has no effect on the enzymes of G. crumenifer, while

ATPase of membrane origin in both the parasites is significantly

inhibited.

Rafoxanide and oxyclozanide drugs belong to the

salicylanilide group. It is interesting that the effect produced

by these drugs show some degree of similarity and dissimilarity

reflecting the variation in the physicochemical properties of the

enzyiTi molecules. Oxyclozanide had an inhibitory effect on Y-GTP

of soluble fraction in G. crumenifer while it produced either

stimulatory or no effect on ATPase, acid and alkaline

phosphatases of the two fractions in both the parasites.

In order to find out the effect of these drugs on 3

transmembranosis, H-glucose uptake was investigated in rumen

parasites in presence and absence of these drugs. Results of

glucose uptake study revealed that mebendazole has a marked 3

inhibitory effect on H-glucose uptake, which was found to be

concentration dependent i.e. inhibitory effect increases with the

increase in drug concentration. While oxyclozanide and rafoxanide 3

had stimulatory effect on H-glucose uptake by rumen parasite G.

•crumenifer.

To monitor the effect of oxyclozanide on the host immune-

system, sera were raised against the soluble fraction of whole

homogenate of G. crumenifer in drug treated and untreated

animals. The sera were tested against the partially purified

8.J

membrane antigens by reversed rocket immuno-electrophoresis. It

was observed that height of the rocket peaks were directly

proportional to the antibody concentration. A gradual decrease in

the height of the rocket peaks corresponding to the antibody

concentration was noticed between the control and drug treated

animals. The overall suppression in antibodies level was found to ft

be 43X in drug treated animal sera. It is therefore concluded

that oxyclozanide has immunosuppressive effect.

8f;

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