- ^

CHARACTERIZATION OF DEVELOPMENTAL STAGE-SPECIFIC POLYPEPTIDES OF SOME

HELMINTH PARASITES

DISSERTATION

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF

iWaj0(ter of ^Ijilosiopljp

IN

Hoologp I

SkK

By -

SABIHA KHATOON

Under the supervision of

DR. MD. KHALID SAIFULLAH

DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY

ALIGARH (INDIA) 2011

DS4188

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J External : 2200920/21 P*'°"®^1_Internal .3430

DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY

ALIGARH-202002 INDIA

Sections: 0. No /ZD 1. ENTOMOLOGY 2. FISHERY SCIENCE & AQUACULTURE Dated: 3. GENETICS 4. NEMATOLOGY 5. PARASITOLOGY

Certificate

This is to certify that the dissertation entitled "Characterization of

developmental stage - specific polypeptides of some helminth

parasites" being submitted by Ms. Sabiha Khatoon embodies original

work done by the candidate herself. The entire work has been carried out

under my supervision.

I permit the candidate to submit the work for the award of the

degree of Master of Philosophy in Zoology from Aligarh Muslim

University.

Dr. Md. Khalid Saifullah (Supervisor)

"In The 'Xame ofALLA'JC The Most (Beneficent, The Most 'Mercifuf. "J4CC

thanl^ to aCmighty JA [[ah for giving me an opportunity, determination and

of course so 'Wonderfu[peop[e around me to comp[ete this research wor^

It is indeed a matter of great p[easure for me to suSmit my dissertation on

"Characterization of deve[opmenta[ stage-specific po[ypeptides of some

he[minth parasites", a topic assigned to me for the partia[fu[fi[[ment of the

M.<Phi[ in Zoo[ogy ((Parasito[ogy)fromJAMV.

I ac^oudedge the h^en interest, superior guidance as we[[ as constant

support By (Dr. Md. %fia[id Saifuttah, my supervisor, throughout this

research xvorh^ Jfis magnanimity, generosity and gracious attitude a[ways

l{ept me afresh during the course of research worh^

I am indebted to (Prof. Irfan ^hmad Chairman, (Department of Zoo[ogy,

JiMV, J4[igarh, for providing me various faci[ities during the research

worh^

I %vou[da[so [i^ to than^my teachers (Prof. 'Wajifiullafi (Section inchage),

(Prof. S.M.A- A^idi, (Dr. MaCi^ IrshaduCCafi for their constant

encouragement and guidance at every step of my research.

Ms. ShaBnam 'Kjian has Seen tfie wondeifu[ senior and is just a ca[[ away

to so[ve any pro6[em I am high[y thanhfu[to her. M^ords are inadequate to

express my genuine thanh^ to my (aS mates JLhmad Shareef M. JL- Tfannan

%fian, Yasir JL^tar ^an andSha^eCAhmadvuho he[pedme out in every

possiShs way. Specia[ thanh^ are due to my friend Ms. Sadia 1(flsfiidfor

[ending me a [ie[ping hand during the most stressfu[ days, despite of a[[ her

Busy scheduhes.

I fa[[sfiort of words to express than^ to my sisters ancffriends Ms. J4nees

yiSSas ^Rjzvi and (DiCnasheen Meerza vufio were afways ready to extend

their sfioufder of support whenever it was required, without their he [p this

worl{jwas not possiSfe.

I must express my thanh^s to Mr. Sha^rJiCi, Mr. ^mesh Chandra and Mr.

yizam and Mr. Satfaraz, for their cooperation and assistance in the sampCe

coCCection. I woufdfih^ to thanh^Mr. ^eesfor his cooperation andheCp.

I talie this opportunity to appreciate and thanh^aCf my juniors, especiaCfy

Jisim (Rjzvi, Mohd. Maroof LuBna, hman andJltifZafar

Specific thanlis to Mr. Va^eC and Mr. %flfee[ for providing me the

chemicals which were needed 6y me.

I owe my deepest thanh^ to the most precious gift ofC^od, my Coving mother

and my Brother Mohammad ^shad for Being supportive in my every

endeavor.

I must also open the truth here By saying that I have succeeded; it is onCy

Because of my teachers of Luchnow Vniversity from where I have done my

post graduation. They supported me in every part of my Cife.

Thanfis are due to aff those peopfe who are directly or indirectCy fin^dto

this worh^ I dedicate this worh^to my teachers from Intermediate to post

graduation.

SaSiha Xfiatoon

m

DEDICATED TO

Y TEACHER

Sk m

CONTENTS PAGE NO.

ACKCOWLEDGEMENT

INTROUCTION 01-07

HISTORICAL REVIEW 8-15

MATERIALS AND METHODS 16-27

RESULTS 28-52

DISCUSSION 53-63

REFERENCES 64-79

MTEODUCTlOl

m

Introduction

India is basically an agricultural country where livestock constitute an

important component of animal wealth. Livestock like buffaloes and cattle provide

meat, milk and other dair}' products which arc enriched sources of proteins in our diet

therefore importance of buffaloes and cattle cannot be overlooked. Among the

developing countries India has a staggeringly high incidence of malnutrition and as a

result major segment of population suffers from poor health that ultimately affects

working efficiency of an individual. Apart from fulfilling nutritional requirement, the

animal wealth contributes substantially to the welfare and economy of rural masses.

Asian buffaloes dominate the world buffalo population, representing 96.4% of

the worldwide population, which is 180.70 million that is 17419.48 million buffaloes

have been reported from Asia (FAO, 2010). Within the Asian region, about 78.8% of

buffaloes are in the South, 12.8% in East Asia, and only 8.4 % are found in South

East Asia. Among the Asian countries India has the largest population (54.562%) of

buffaloes. According to FAO (2008) buffalo milk production in Asia represents

96.78% of total volumes of 89.2 million tons of World's buffalo milk. Buffaloes are

significant source of milk and contribute 56.85% in total milk production in India. In

addition to dairy products, buffaloes contribute to our leather industry, transport as

well as serve as a source of fuel and fertilizers. According to National accounts

Statistics-2011 (central statistical Organization; Government of India), agriculture

shares 15.33% and livestock shares 3.93% in gross domestic production (GDP).

According to department of Animal Husbandry, Dairying and Fisheries, Ministry of

Agriculture, Government of India (GOI) 2010, total production of milk in India is

112.5 million tormes and per capita availability is 263 (gms/day) while in Uttar

Pradesh it is 2,0,203 tormes.

According to National Accounts Statistics-2011, Central Statistical

Organization, Government of India (GOI), it was estimated that during 2009-10 that

income generated from livestock sector by milk group is Rs. 2, 28,809 crores, by meat

group is Rs. 64,073 crores and the total value of output from livestock sector is Rs. 3,

40,473 crores. The dairy products are important sources of nourishment. Buffaloes

contribute 5?^% of the milk though they constitute only 30% of the total bovine (cattle

and buffaloes) population (Acharya, 1988)

Considering the vital role of cattle in our economy and their contribution to national

income, the government has paid attention to the development of livestock in general

and buffaloes in particular. Now, there is a rich genetic diversit> available in India for

all the species of livestock. We have 26 breeds of cattle, 7 breeds of buffaloes, 20

breeds of goats and nearly 48 breeds of poultry, contributing to our gross domestic

product (GDP). According to 2007 census, India has about 199.1 million cattle, 105.3

million buffaloes, 71.6 million sheep, 140.5 million goats and 11.1 million pigs.

In tropical and sub tropical countries, the importance of buffalo for food,

energy, leather and agriculture is well recognized. Despite its importance very little

attention has been paid to improve buffalo health, although in many Asian countries

national development programme have been initiated to develop genetically improved

and highly productive buffalo breeds. Increased production can only be achieved if

proper buffalo health management programmes including the parasitic diseases are

planned and effectively implemented (Nizami, e( al, 1991). Milk and meat

production and draught animal power can be improved through selective breeding and

effective animal management. However, meaningful results can only be obtained if

equal attention is paid to the health of animals.

Despite their importance in country's economy, the net contribution made by

India's animal wealth is not much. The poor animal health has been considered as one

of the major factor for meager contribution of livestock to the national income. A

wide array of helminth parasites beside viral, bacterial and protozoan mfections

causes heavy morbidity and also mortalities during the epidemic outbreaks. In order

to prevent losses incurred by such parasitic menace, it is essential that we should

protect our farm animals from these infections.

The World health organization (WHO) estimates that roughly half of the

world population and much of its livestock are infected with parasitic helminthes.

Half a billion people mostly living in developing countries suffer devastating,

sometimes fatal illness as a result of these infections. Moreover the effects of

helminth parasitism may be dangerous and may bring about significant cognitive

deficit in untold millions. Helminth parasites erode livestock production often most

severely in already femine prone areas with marginal agriculture. In India helminth

infections are very common in all the varieties of animals. Faciolosis, schistosomiasis,

paramphistomosis etc. are most important helminthic infections affecting our farm

and domestic anmials.

Amphistomes constitute one of the most common groups of digenetic

trematodes of domestic livestock. They are distinguished from other trematodes by

the possession of a posteriorly located acetabulum. Infections by adult amphistomes

may be commonly found in sheeps, goats, cattle and water buffaloes in India as well

as in other tropical countries. The disease "paramphistomosis" is caused by massive

Infection in small intestine with immature paramphistomes and this disease is

characterized by acute gastero-enteritis with high morbidity and mortality rates,

particularly in young stocks. Prevalence of infection in some localities reaches up to

90%. Magnitude of parasitic infection in livestock drastically affects Indian agronomy

and therefore requires effective control measures.

Adult paramphistomes occurring in the rumen are generally considered to

have a low pathogenicity, while heavy bile duct infections cause superficial

haemorrhage, pronounced periductal fibrosis and other hyperplastic changes (Kulasiri

and Senevirante, 1956; Arora and Kalra, 1971; Jha et al, 1977,) the migrating

immature paramphistomes in the intestine cause severe pathological changes. The

usual picture is of acute catarrhal and haemorrhagic inflammation in the abomassum,

duodenum and jejunum with associated anaemia, hypoproteinaemia and oedema. The

symptoms of the disease include general v/eakness, increasing anorexia and

polydypsia.

More than 70 species of amphistomes have been described in cattle host (Sey,

1991) and approximately 40 species of amphistomes have been reported from India

(Agarwal, 2003). The prevalence and intensity of infection with these species have

been reported in ruminants throughout India (Dutt, 1980). Survey from our laboratory

on the prevalence of amphistomosis revealed, 71.4% of rumen buffaloes were found

infected with different species of amphistomes with varying intensities of infection,

with Gastrothylax crumenifer (32.7%), Fischoederius elongatus (6.0%),

Pcn-amphi'itomum epicUtum (51.9%), Orthocoelium scoliocoelium (16.1%). while in

liver Giguntocoiyle explanatum infection was observed in 19.6% animals (Hanna el

a!., 1988). Besides large visible losses through mortality of young, sick and

malnourished animals, there are undoubtedly huge economic losses resulting from

sub-optimum productixity, due to clinical and sub-clinical infections.

Considerable work has been done in India to tackle this problem. Initial attention was

paid on taxonomy, morphology, life cycle of parasite, pathogenicity and treatment

were dealt by different workers ((Bhatia and Chauhan, 1984; Chwodhury et al,

(2002); Sood (2003a); Tandon and Dhawan (2005); Haque et al, (2011). Biochemical

and immunological aspects are emerging areas attracting attention of Indian

Scientists. Studies relating to biochemical, physiological and immunological aspect of

amphistomes in Indian perspective are very little. A number of studies by Khan et al,

(1990), Abidi and Nizami (1995), Saifullah (1999), Ahmad et al, (2004) and

SaifuUah et al, (2011) are available.

The life histories of numerous paramphistome species have been viewed by Yamaguti

(1975). The life cycle (Figure, 1) of those parasitizing rumen and bile duct involves a

sexual phase of reproduction in the definitive host and an asexual phase in a

moUuscan intermediate host. The adult parasite is found in the fore stomach, bile

ducts or intestine of the definitive host, depending on the particular species. Large,

non-embryonated and operculated eggs possessing a peculiar keratin - type shell

(Madhavi, 1966; Arfin and Nizami, 1986) are voided with the faeces and if they fall

in water, development is completed in 7 to 10 days during warm weather.

Gastrothylax crumenifer miracidia takes longer time to develop than Gigantocotyle

explanatum. Most of the studies have been done on adult amphistome parasites while

the developmental aspects have been neglected and therefore needs attention. Present

study was focussed on the miracidial development of paramphistomes, Gastrothylax

crumenifer and Gigantocotyle explanatun. Upon hatching, the free-swimming

ciliated miracidium must locate and penetrate a suitable molluscan intermediate host

within 8 to 10 hours. Numerous and diverse snail species have been found to act as

intermediate hosts for larval paramphistomes (Monning, 1938; Soulsby, 1971) but in

India, the commonly infected species are Indoplanorbis exustus, Gyrallus

convexiusculus and Lymnaea sp. (Firasat and Nizami. 1989). The miracidium

Figuiel: General life cycle of amphistome parasites.

1: Buhalis huhalis (Definiti\e host).

2: Adult amphistomes 2A: Gaslrothylax crimemfer 2B: Gigantocotyle explcmatum 3C: Epiclitum explanalum.

3: Freshl} hatched egg.

4: 4-day egg.

5. hgg containing mature miracidia (hm).

6: Miracidia emerging from egg shell.

7: Full} hatched free swimming miracidia,

8: Intermediate host snail.

9- Sporoc^st

10: Rcdia.

1 1; C'ercaria.

12: Metacercaria.

I Figures 3. 4. 5, 6 and 7 are adapted from Tandon (1957) and Figures 10 and 11 are adapted from Wille\ (1936).

Figure 1: General life cycle pattern of Amphistome

parasites.

transforms into a small, short lived sporocyst in the foot, mantle or tissue lining the

respirator)- chamber Numerous asexually produced rediae are liberated, which

migrate through the snail tissues to the digestive gland tissues before emerging from

snail. Mature cercariae are large and often highly pigmented having distinct eye spots.

They are encysted on vegetation and shed theii tails and ultimatel) form metacercaria!

cysts. When consumed by a suitable host, the metacercariac cxcyst in the duodenum,

either penetrate the mucosa or sometimes the sub mucosa where they feed for several

weeks on host tissue before migrating to their final site of development in the rumen,

bile ducts, caecum or colon.

Infected animals are rarely treated with anthelmintic for adult

paramphistomiasis. The removal of adult parasites can be of prophylactic significance

as it reduces the reservoir infection for intermediate hosts. Several anthelmintics have

proved to be effective against immature stages in ruminants (Boray, 1986). The

literature available on various aspects of amphistomiasis is mostly confined to cattle,

sheep and goats and there is a scarcity of data concerning buffaloes.

The devastating situation created by paramphistomosis requires a control

measure to save livestock. Before the formulation of any chemotherapeutic means, it

is important to understand the parasite biochemistry and physiology, host parasite

relationship, diversifications of biomolecules and adaptation found among different

groups of parasitic helminthes. Knowledge of these aspects will be usefiil to explore

the various mysteries which can be exploited rationally to control the parasitic

problem. Although a tremendous literature relating to parasite biochemistry and

physiology of trematodes has been reviewed by many workeis (Goil, 1957, 1958a,

b;Yusufi and Siddiqi, 1976; Bahadur and Gupta; 1984, von Brand; 1979, Barrett,

1981; Smyth and Halton, 1983; Khan et al, 1990; Abidi and Nizami 1995; Saifullah

and Nizami, 1999), the branch of helminth physiology and biochemistry is still in

developing phase. Understanding the physiology and biochemistry of a parasite in its

original habitat is a difficult task. Amphistomes like all other parasites complete their

life cycle in different environment and experience the challenges posed by different

micro and macro habitats . In micro habitat, a parasite has to struggle for their

existence hy adapting various physico-chemical conditions as a result a regular

sequence of metabolic switches occur during the life cycle of parasitic helminthes.

There are various metabolic adaptations which distinguish parasites from their free

living counterparts and give them a unique character. Apart from studying

physiology, sound knowledge of parasite biochemistry is required for the

development of new drugs and effective vaccines. The biochemical peculiarities

identified in the parasite and host can be conveniently exploited for the application of

chemotherapeutic and inimunological control measures. Thus parasitic idiosyncracies

evoked the interest of investigators to unravel the unique targets possibly occupying

during the course of larval development.

The aim of the present study is to investigate stage -specific polypeptides and

their characterization, to determine total protein content, to find out enzyme turn over

and to resolve isozyme profile of GST and proteases during the miracidial

development of Gastrolhylax crumenifer and Gigantocotyle explanatum. Further, the

biochemical peculiarity during this study may be helpful for the development of

control strategies to prevent the spread of infection to ruminants.

w

ISTORICAL

EE^ffiW

&

Historical review

The developmeulal stages of amphislome parasites are characterized by a

striking morphological and biochemical transitions between an intermediate

moUuscan host, living in an aquatic environment and warm blooded mammalian host,

where they undergo multiplication for genome amplification and become sexually

mature. They represent an ideal but challenging biological system. The regulatory

activities and response of the parasites to the environmental changes are manifested at

the morphological, reproductive and even at the molecular level for improved survival

strategies to ensure the establishment of their germ line. The complex life cycle of

parasites involve a regulatory sequence of metabolic switches regulated by required

alterations of biomolecules by the parasites. Thus, any change in the organic state of

the habitat or its physico-chemical condition is reciprocated by the parasites through

its metabolic features.

Survey of the literature reveals that most of the studies like morphological,

physiological, biochemical etc. are confined only to adult parasites while other

developmental stages particularly free living stages and those infecting the

intermediate moUuscan host are almost neglected. Among the developmental stages

of helminths most of the studies have been restricted to Schistosomes, Fasciola spp.

and Hymenolepis spp., as evident from a number of reviews available on the parasite

biochemistry and physiology (Smyth, 1966; von Brand, 1979; Chappel, 1980; Barrett,

1981; Cox, 1982; Smyth and Halton, 1983; Arme and Pappas, 1983). Among the

nematodes, eggs of Ascaris spp. have been largely used for most of the metabolic

studies. The energy metabolism has been widely studied in order to investigate the

possibilities of chemicals which could be exploited for chemotherapeutic control

(Cheng, 1963; von Brand, 1973; 1979). It is generally accepted that whenever cellular

differentiation is involved, protein and nucleic acid play a vital role. According to

Smith and Walker (1986) nucleic acid synthesis is followed by protein synthesis in

cellular differentiation in gonads of Asterias vulgaris. The eggs of Schistosoma spp.

have been reported to be rich in enzymes (Pepler, 1958, Andrade and Barka, 1962).

The protein content of different developmental stages of Nippostrongylus

brasiliensis was found to differ considerably and constituted about 76% of the dry

weight of iu\'pnile.s. (Fiorkin and Scheer 1969). Though protein synthesis proceeds at

a high lale m trematodes, only a few studies ha\e been carried out on their larva!

stages. The hiochemical composition of developing eggs of Fasciola spp. amino acid

metabolism in sporocysts, rediae and cercariae of Glypthelmiiis spp., Gorgoderina

amplicuna and Echinoparaphum spp. have been mvestigated (Cheng, 1963 and

Wilson, 1967).

Wilson (1967) used histochemical and physiological tecliniques to investigate

the changes in biochemical composition associated with the development of miracidia

and reported a sharp decline in the gross protein during the cellular organization of

miracidia. Analysis of protein components has been performed by various workers by

using acrylamide gel electrophoresis (Sodeman. 1967; Kusel, 1972; Kusel and

Mackkenzie. 1975; Murrell et ai, 1974; Pelley et al, 1976). The SDS-PAGE results

revealed up to 60 bands in each sample and most of the bands were of similar

intensity in immature and mature forms of male and female of 5". mansoni. It was also

reported that a protein with high molecular weight (160 kDa) was unique to cercariae;

bands with 58 kDa, 23 kDa and llkDa were unique to adult Schisotsomes while

protein of 29 kDa was unique to female worms (Ruppel and Cioli, 1977). The authors

also observed high degree of identity between protein pattern of immature and mature

Schistosomes and between male and female worms. Inspite of great morphological

differences and specialized structures, characteristics of each stage are likely to

emerge as differences in protein composition only, if they represent a sizeable portion

of the total parasitic tissues. A few prominent bands have been observed to be present

only in specific developmental stages. Thus, a protein of 160 kDa was unique to

cercariae and that might be associated with either glycocalyx which was absent in

post cercarial stage or with the contents of the penetration glands which were

discharged during skin passage. Adult worms of both sexes possessed a protein of

about 58 kDa which was lacking in immature stages and might therefore be associated

with appearance of some typical adult structures, like the intestine and its contents.

The same is true for the other two proteins from adult worms of about 23 kDa and 11

kDa. Further, a protein of 29 kDa occurred only in female worms and is likely to be

associated with the female genital organs (probably the vitelline glands which occupy

the greater part of the female body). Female- specific proteins were also described by

Ruff el al (!971) for .S" japomciim Electrophoretic anahsis by SDS-PAGE of

soluble egg proteins of 5. mamom. (Carter and Colley. 1978; Pelley and Pelley 1976;

Pillay 199S 1996). G crumenifer. F elongatus, P. epiclitum, O. scoUocoelhim and G.

explanatum, F. gigantica (Saifullah and Nizami. 1994) has also reported and the data

have been utilized tor taxonomic studies. Saifullah and Nizami, (1994) identified a

total of 52, 50, 44. 67, 52 and 41 polypeptides with 3, 5, 4. 14, 4 and 9 charecteristic

polypeptides in the freshly collected eggs of G. crumenifer, Fischederus elongates, P.

epiclitum and Orthocoelium scoliocoelium, G. explanatum and F. gigantica

respectively. - differences between adult worm and schistosomula have also been

reported (Ruppell and Cioli, 1977; Shah and Ramasamy, 1982; Maeda et al, 1984)

analysed the protein composition of S. japonicum (excretory/secretory) products,

soluble extract of newly laid eggs in vitro matured eggs by SDS-PAGE. These authors

observed that the silver staining pattern of newly laid eggs and mature eggs were

similar except for the band at 66 kDa which appeared in highest concentration in

newly laid eggs. The soluble egg antigen of Schistosoma japonicum was fractionated

in to 20 individual isoelectric point fractions with pi ranging from 1.86 to 11.40 by

lEF. Fractions in the acidic region may play an important role in elicitation in

schistosomiasis (Zhu et al., 1994)

General metabolism of egg is poorly known and has been examined only in

few species. Studies on biochemical turnover in larval helminthes reveal that the

endogenous reserves are utilized during the development of miracidia of Fasciola

hepatica and G. explanatum (Hortsmann, 1962; Wilson, 1967; Khan et al, 1991). In

the eggs of F. hepatica a decline in protein content during the development (0-10

days) was observed. The protein content at the begirming of development was

recorded as 80-100 ^tg/1000 eggs which decreased to 20-30 ig/lOOO eggs. This

decrease in soluble protein could indicate either combustion as a food source or

combination in to structural components as observed by Wilson (1967) who also

found that carbohydrate and protein appear to be the main energy source in a ratio of

3:2. Further, the contribution of shell material to total dry weight (20%) is

considerable and appears to be predominantly protein while most of the remaining

80% of egg solids can be accounted for 27% protein, 26% carbohydrate and 10%

lipid.

10

I ike other metabolites, the trematode eggs are also rich in enzymes which

participate in developmental regulation of the organism. A number of enzymes like

acid and alkaline phosphatases, aminopeptidases. acetylcholine esterase and a non-

specitic esterase have been detected in eggs of .S'. mansoni (Andrade and Barka, 1962.

Pepler, 1958). The isozymes of eight enzymes of Schistosoma cecariae have also been

investigated by Boissenzon and Jelues (1982) whereas; Proberl (1966) reported

various enzymes from redia and cercaria of Echinoparyphium recurvatum. Thus the

presence of large enzymes reflects the importance of protein metabolism.

A lot of work has been done on carbohydrate metabolism, but only limited

work has been carried out on the protein metabolism on trematode developmental

stages. Protein synthesis undoubtedly proceeds at a high rate for larvae to build not

their own tissue but also of those of the individuals of subsequent generation. The

origin of the amino acid involved in the synthesis of protein in rediae, sporocysts or

cecariae has been examined in a number of species, e.g Glypthelmins spp Gorgodera

amplicava and Echinoparyphium spp. (Cheng, 1963)

A number of key enzymes belonging to the group transaminases,

oxidoreductases and deaminases have been investigated in some helminth parasites

(Barrett, 1981). The most important of these enzymes include glutamate

dehydrogenase (GLDH), Aspartate transaminase (GOT), Alanine transaminase

(OPT), ornithine transaminase, y-Glutamyl transpeptidase, arginase, monoamine

oxidase (MAO), acid and alkaline phosphatases, proteases and some antioxidant

enzymes.

Transaminases: The transaminases comprise a group of enzymes which are

involved in the transfer of a amino group of a number of amino acids. These enzymes

also serve as clinical markers when investigated in the serum of the infected

individuals, and at times the increase in serum level of the host may partly be

contributed by the parasites (von Brand 1979; Barrett 1981; Smyth and Halton; 1983).

Therefore, the parasite transaminases may also be of considerable interest. Although

transaminases were identified in animal tissues as early as 1937, very few

experiments have been performed on these enzymes in parasitic helminths. Daugherty

11

(1952) found that homogenates of the liver fluke. Fasciola hepatica. contained very

high transaminase acti\ ity.

Among helminthes the transammases have been nivestigated m a number of

trematodes, cestodes and nematodes (Garson and WilHams. 1957; Min and Seo, 1966;

Connolly and Downey 1968; Sturn et al. 1972; Tandon and Misra, 1984. The activity

of 2-Oxoglutarate transaminases has also been investigated in F. hepatica (Park et al,

1983) in S. cervi (Lee et al, 1983), in A. srivastava (Singh et al, 1983) in A. suum

(Dubinsky et al 1984, Zenka and Prokopic, 1983) and in Nippostrongylus brasiliensis

(Watts and Atkinson, 1983, 1984). Alanine and aspartate transaminase activities were

also reported from redia of Crypyocotyle lingua and sporocyst of Cercaria emasculus

(Watts, 1970).

Phosphatases: Phosphatases have been demonstrated in a number of helminth

parasites where they are considered to play an important role at the transporting

surfaces (Lumsden, 1975; Prashad and Guraya, 1977). These enzymes have been

studied in parasites such as Acanthocephala (Bullock, 1958), Ancylostoma caninum

(Murakami, 1961), Ancylostoma duodenale (Marzullo et al, 1957) Ascaridia galli

(Marsh and Kelley, 1958), Ascaris lumbricoides (Thanaka, 1961, Chowdhury et al,

1955), Cysticercus tenuicollis (Erasmus, 1957a), Dipylidium caninum and F.

hepatica (Tarazona Villas, 1958), Haemonchus contortus (Marsh and Kelley, 1958),

Hymenalepis nana (Gilford, 1957), Moniezia expansa (Erasmus, 1957b), Schistosoma

mansoni (Dusanic, 1959, Robinson, 1961) Taenia pisiformis (Erasmus, 1957a),

Taenia saginata (Chowdhury, 1955), Wuchereria bancrofti (Chowdhury et al, 1955).

Phosphatases of four helminth species, Ascaridia galli, Cotylophoron cotylophorum

and Raillietina cesticillus have been analysed colorimetrically and also the effects of

pH and various chemicals and anthelmintics on the enzyme activities were analysed

by Prashad and Guraya in 1978. Histochemical observation of alkaline phosphatase in

S. mansoni adults, and its developmental stages (eggs, miracidia, sporocysts and

cercariae) was done by Dusanic in 1959. Two bands of isozymes of alkaline

phosphatase activity were found in adults and cercariae by Pino et al, (1968).

Comparative histochemistr>' of alkaline and acid phosphatase in mature and immature

F. hepatica was done by Thorpe in 1968 and low levels of alkaline phosphatases were

observed in immature than mature parasite. Changes in acid phosphatase activity were

12

reported in developing and aging Nippostrongylus brasiliensis by BoUa et al., (1974)

and the pH optimum and maximal enzyme activity was found to be correlated with

the developmental stage and age of the parasite. Alkaline phosphatase activity in the

soluble egg protein of Schistosoma mansoni was observed (Cesari et al., 2000).

Isozymes of alkaline phosphatase were identified in daughter sporocyst of S. mansoni

(Ballen et al, 2002).

Proteases: Proteases hysrolyze peptide bonds. On the basis of important chemical

groups in their active sites, proteases are separated in to major classes: serine,

aspartic, metallo and cystein proteases. Proteases catalyze a broad spectrum of

important biological reactions leading to activation of enzymes, hormones and peptide

trophic molecules. These enzymes are involved in blood coagulation and fibrinolysis,

protein metabolism, immune reactions, and tissue remodeling (McKerrow, 1989 and

Tort et al, 1999). Metallo- and serine proteases are known to act in tissue /cell

invasion process in parasitic organisms, but recently cystein proteases have been

implicated in invasion by many helminth parasites (Sajid and Mckerro, 2002).

Proteases released by Ostertagia ostertagi, a bovine abomasal nematode, have been

found to degrade bovine mucin (Geldhof et a!., 2000). The excretory/secretory

products of the infective stage larvae of Trichinella spiralis are reported to contain

serine, aspartic and cysteine proteases (Criado et al, 1992, Moczon and Wranicz,

1999, Lun et al, 2003) and zinc dependent metalloproteases, with a substantial

contribution of metalloproteases, {Lun et al, 2003). Proteases also play important

roles in digestion of food (Stocker and Zwilling, (1955), hatching of egg (Yasumasu

et al, 1992) and in hydrolysis of extracellular matrix component (Yan et al, 2000).

Serine and metalloproteases are reported to be present in excretory/secretory products

of microfilariae and adult males of Onchocerca vovulus (Haffiier et al, 1998). The

cathepsin L-like proteases are secreted in gut of F. hepatica (Halton, 1997). A cystein

protease located in the cercarial penetration glands of Diplostomum pseudopathaceum

is thought to be involved in skin penetration of aquatic birds (Moczon, 1994). A very

interesting study of Fishelson et al, (1992) elucidated how the parasitic blood fluke 5.

mansoni synthesizes, stores and releases a serine protease during differentiation of its

invasive larvae. Metalloproteases are reported to be present in migrating larvae oi

cestode Protocephalus amhloplitis parasitic in fishes, has collagenolytic.

1-?

haemoglibinolytic and slight elastinolytic activity (Polzer et al, 1994a). In the cestode

Schistocephalus soUdus parasitizing birds, a chymotrypsin- like protease with

collagenolytic activity was present in procercoids but not in plerocercoids or adults,

possibly being necessary for the penetration of host intestinal wall (Polzer and

Condrat, 1994b). In vitro Studies have demonstrated that oncosphere of Hymenolepis

diminuta release a serine protease from penetration glands just after emerging from

their embryophores (Moczon, 1996). It has been documented that excretory secretory

products of Spirometra mansonoides plerocercoides contain tissue lysing enzymes:

cysteine protease of 21 KDa and 28 kDa, trypticases of 104 and 198 KUa, as well as

chymase activity corresponding to 36 kDa (Cho et al.. 1992, Song and Chappel,

1993). Pino-Heiss et al., (1985) reported that both eggs and miracidia of Schistosoma

mansoni secrete proteases which are capable of degrading at least the glycoprotein

components of extracellular matrix to facilitate their migration through intestinal wall

or penetration of snail host tissue. Mohamed et al., (2005) assessed the proteolytic

activity of 0-12 day old eggs, miracidium and adult worm of F. gigantica. The results

appeared that the proteolytic activity increased during embryogenesis followed by

miracidium and adult. The proteases of both microfilariae and L3 larval lysates of

Brugia malayi were analysed by SDS-PAGE using gelatin copolymerized gels which

showed complex array of proteases with varying molecular weights (Bhandary, 2006).

In recent studies proteases are used as a drug target and vaccine development.

Glutathione S-transferase (GST): GST is an antioxidant enzyme which is

involved in xenobiotic metabolism, intracellular binding, and in biosynthesis of

endogenous substrates, such as prostaglandins and leukotriens (Boyer, 1989).

Glutathione S- transferase isozymes which have very little homology with those from

mammalian tissues have been found in helmiths, such as trematodes and nematodes

(reviewed by Rao et al, 2000). Immunohistochemical studies revealed that

glutathione S-transferase in worms are predominantly associated with metabolic and

reproductive sites of the organism (Rao et al, 2000). GST of helminth origin are

implicated in a variety of metabolic pathways by conjugation of glutathione to

different agents, mcluding detoxification of anthelmintic substances and intracellular

transport (Precious and Barrett, 1989; Brophy and Barrett, 1990; Sharp et al, 1991;

O'Leary and Tracy, 1992; Miller et al. 1994; Kampotter et al. 2003; Leirs et al,

14

2003). Studies related to GST have been carried out in S mansom, S japonicum and

F hepulica (Gupta and Srivastava, 2006). Native and recombinant GS1 s have been

characterized in Haemonchus contortus (Sharp et al, 1991). In 7" spiralis glutathione

S- transferase functions as selenium independent glutathione peroxidase was detected

by electron microscopy in the secretor)' organs (stichoc\tes) of LI larvae which

played a protective role against lipid peroxidation (Rojas et al, 1997). In filarial

nematodes the presence of selenium-independent glutathione peroxidase activity

associated with nematode glutathione S-transferase has been described in D. immitis

(Jaffe and Lambert, 1986). In adult O. volvulus, one of the two identified isozymes of

glutathione S-transferase has been found to be released by the worms (Liebau et al,

1994). Glutathione S-transferase was found to be ubiquitous in all stages of Brugia

phangi and B. malayi life cycle. A high activity of this enzyme was observed in L3

and L4 larvae (Rao et al, 2000). GST has been reported to be present on the body

surface of the trematodes S. mansom (Taylor et al., 1988) and F. hepatica (Wijffels et

al, 1992). Oesophagostomum dentatum dexelopmental stages were investigated for

GST expression at the protein and mRNA level by Joachim et al, (2008) and it was

reported that the GST activity was present in all the developmental stages (Infectious

and parasitic stages including third and fourth larvae of different ages as well as males

and females). Different GST isoforms have also been characterized for A. suum

(Liebau et al, 1994), H. polygyrus (Brophy et al, 1994) and O. volvulus (Liebau et

al, 1994).

From the survey of literature it is revealed that biochemical peculiarities of

amphistome parasites specifically developmental stages are not worked out. The

present study may be helpful to discover and develop the innovative strategies of

parasite control.

ATERIA: r ^ /

METHODS

i

Materials and methods

Collection of adult parasites:

The adult amphistomes, Gastrothylax crumenifer and Gigantocotyle

explanatum infecting rumen and liver of Indian water buffaloes {Buhalns bubalis)

respectively, were collected from local abattoir (Figure, 2). The fresh worms were

brought to laboratory very quickly and carefully. Parasites were washed 3-4 times

with Hanks' balanced salt solution (HBSS) without glucose premaintained at 37±2 °C.

After washing, the worms were dried on filter paper and stored in vials at -20 ''C until

use.

Collection of different developmental stages of eggs:

Mature and active parasites were washed and incubated in Hanks' balanced

salt solution (HBSS) premaintained at 37 ±2 ^C in beakers for 12 hrs to collect fresh

eggs. After every 4 hrs medium was changed to avoid turbidity and toxicity due to the

released excretory/secretory products. After completion of incubation the parasites

were removed and the eggs settled at the bottom, were washed with tap water 3-4

times and finally with distilled water. Some of the freshly laid eggs (0- day eggs.

Figure, 3A) were stored at -20 ' C for future studies. Rest of the egg samples were

equally distributed in two different beakers containing tap water. During the

incubation (at 25 °C), water was changed regularly and aeration of eggs was done

everyday 3-4 times. After every 24 hrs the eggs were inspected daily under light

microscope to moniter miracidial development in eggs. Eggs were taken out from one

beaker after four days (4-day eggs, Figure, 3B) of incubation and stored at -20 °C.

Second set of beaker containing eggs was left until mature wriggling movement of

meracidia was observed inside the eggs. Mature miracidia along with egg shell (Em,

Figure, 3C) were collected and stored at -20 °C for further study.

Preparation of homogenate:

For protein profile and enzyme assays, a 10% homogenate of eggs and adult

parasite was prepared using 0.1 M phosphate buffer (pH 7.4) in a Teflon tissue

homogenizer. Homogenate was centrifuged (Hitachi Koki Co., Ltd. Tokyo Japan) at

the 6000 xg for 10 minutes at 4°c. After centrifugation, the supernatant was collected

and immediately stored in aliquots at -20 °C.

16

Figure 2: A- Infected liver showing heavy infection of liver amphistome Gigantocotyle

explanatum

.0

B- Infected rumen showing infectiji of rumen amphistome Gastrothylax crumenifev.

B

F i g u r e 2 : Showing infected liver (A) and rumen (B).

17

Protein Estimation:

Protein was estimated b> d^c binding method of Bradiord (1976) as modified

by Specter (1978). The dye reagent consists of 0.01% (w/v) CBBG-250, 5% (v/v)

Ethanol and (10% v/v) Orthophosphoric acid. Bovine serum albumin (BSA) was

prepared in O.IN NaOH as standard protein and the protein, which Vvas measured in

total assay volume 2.1 ml, at a wavelength of 595 nm.

Polypeptide profile by Sodium dodecyl sulphate polyacrylamide gel

electrophoresis (SDS-PAGE), protease profile by gelatin substrate zymography

and isozyme profile of Glutathione S-transferase by native PAGE :

STOCK SOLUTIONS: (Laemmli, 1970)

1. Acrylamide/Bis-acrylamide (30% T, 2.67% C)

Acrylamide- 29.2g/100ml DDW

N'N'-bis-methylene-acrylamide-0.8g/100ml DDW

2. 10% (w/v) SDS (for SDS-PAGE and zymography).

3. Lower gel buffer: 1.5M Tris HCl (pH-8.8) was prepared and the pH was

adjusted with 6N HCl, filtered with Whatman no.l filter paper and stored at

4°C. It was prepared for separating gel

4. Upper gel buffer: 0.5M Tris HCl (pH 6.8) was prepared and the pH was

adjusted with 6N HCl, filtered with Whatman no.l filter paper and stored at

4°C. It was prepared for stacking gel.

5. 1X Running Buffer- 1000ml

Tris buffer - 3.03g

Glycine - 14.4g

SDS - Ig (not in Native PAGE)

DDW - 1000ml

19

6. Ammonium per sulphate (APS): Fresh iO% amonium per sulphate solution was

prepared

Gel formulations for SDS PAGE: Tor SDS-PAGE 10% separating gel and 4%

stacking gel was prepared

GfJ forjjjujatjon for lymogaphy: SimiJarJy, for zymography, J 0% separating gd

containing 0.1% gelatin and 4% stacking gel was prepared.

Gel formulation for GST enzjme or native PAGE: For native PAGE, 8%

separating gel and 4% stacking gel was prepared.

Gel casting:

For SDS- PAGE and gelatin substrate zymography 10% separating gel was

prepared while for GST analysis native PAGE was run using 8% separating gel. The

solution was poured into the mould in between two glass plates which were separated

b} 1 mm thick spacers and held together by clips on a stand. A distance of about 2cm

was left for stacking gel. After complete polymerization, a solution of 4% stacking gel

was poured over the resolving (separating gel) gel. A comb measuring about 1mm

thickness was inserted inside the stacking gel immediately and allowed to polymerise

at room temperature. After polymerization, comb was removed and the wells were

rinsed with distilled water to remove unpolymerised gel, if any.

Sample preparation: Laemmli's Sample buffer (10 ml) - For SDS-PAGE.

Deionised water 3.55 ml

0.5M tris-HCL buffer, pH 6.8 1.25 ml

Glycerol 2.5 ml

10%(w/v)SDS 2 ml

0.5% (wJv) hromomphenol Wue 0.2 ml

50 )il p-mercaotoethanol was added to 950 |il sample buffer prior to use.

Samle and sample buffer were mixed in 1:1 ratio and incubated at 95 ' C for 7-8

minutes. In each well 60 )ig protein was loaded.

Sample preparation for gelatin substrate zymography:

The composition of sample buffer was same as mentioned for SDS-PAGE,

oniy \) mercaptoetnanoi was not auucu anu the sample was mixcu witu sampic buiier

in 1:1 ratio, heating was not done and sample was kept at room temperature only for

15-20 minutes. In each well 10 \xg protein was loaded.

Sample preparation for native PAGE:

The P-mercaptoethanol and SDS were not added in the sample buffer for

native PAGE to avoid reducing and denaturing conditions. In each well 40 |ig protein

was loaded.

Electrophoretic condition:

Soluble protein samples as well as standard protein marker were loaded in

separate wells and the electrophoresis was carried out on vertical slab gel system

(ATTO assembly, ATTO Corporation, made in JAPAN) at 4 "C using a constant

voltage of 100 volts until marker tracking dye reached approximately 1 cm above the

bottom of the gel. After completion of electrophoresis, protein bands were visualized

either by coomassie brilliant blue R-250 (CBBR-250) or silver staining in.

Coomassie Brilliant Blue R-250 (CBBR-250) staining:

The electrophoresed SDS- PAGE gel was washed carefully in deionised water

and fixed in a fixative solution consisting of 10% (v/v) acetic acid, 45% (v/v)

methanol for 2 hr and then stained with 0.25% (w/v) CBBR- 250 dye prepared in

fixing solution for overnight. The ovestained gels were destained in high desatining

solution (HDS), consisting of Methanol: Water: Acetic acid in 45:45:10 ratio for 20-

30 minutes and this was followed by immersion of gel in low destaining solution

(LDS) which consisted of 7% (v/v) acetic acid and 5% (v/v) methanol. Each

Coomassie stained gel was scanned for further analysis.

SILVER STAINING:

I o visualize the protein bands b> silver stain the method of Blum cl al (1987)

was followed. According to this protocol following solutions were prepared for silver

staining.

Fixing solution (100 ml) - Fixing solution consist of methanol (50%), glacial acetic

acid (12%) and formaldehyde (0.0185%).

Sodium thio sulphate (Na2S203) stock solution (0.1%): It acts as a sensitizer and

catalyzes the formation of metallic silver deposits on to proteins.

Pretreatment solution:

Distilled water 99.2 ml

Na2S203 (stock solution) 0.8 ml

Staining solution: The staining solution consists of 0.2%) silver nitrate and 0.03%)

formaldehyde (the formaldehyde supplied with a concentration of 37%o was used).The

solution was prepared fresh and kept in dark brown bottle.

Developing solution (100 ml):

Distilled water 99.93ml

Sodium carbonates 6.0 gm

Na2S203 stock solution 4 ^g/ml

Formaldehyde (37%) 0.05 ml

Stopping solution: To stop the reaction 50%o methanol and 12%) glacial acetic acid

solution was prepared.

Staining procedure:

Gel was fixed in the fixing solution for 1 hour and the gel was washed in 50%)

ethanol twice followed by washing in (30%o) ethanol for 20 minutes. Pretreatment was

done exactly for 1 minute in pretreatment solution and then the gel was washed with

distilled water. Following a quick wash, the gel was impregnated in silver solution in

22

dark for 20 minutes and then rinsed with distilled water and after that the gel was

treated with developing solution for approximately 10 minutes until protein bands

appeared. Then the gel was washed twice in distilled water to remove stain from the

gel and carefully visualized the gel. After thorough washing the gel was treated with

stop solution for 10 minutes. Finally the gel was washed m 50% methanol for 20

minutes and kept it in the distilled water to take photograph.

Double staining procedure: For double staining, the method of Dzandu et al. (1984)

was followed. In brief, individual electrophoressed gel was first stained by silver stain

and then with Coomassie brilliant blue R-250 stain.

Development of zymogram of proteases:

The zymogram for proteases was developed following method of Heussen and

Dowdle, (1980). After the electrophoresis the gel was washed with distilled water

thoroughly. Then the gel was washed in 2.5% (v/v) triton X-100 for at least 3 hr (with

4-5 changes) on a metabolic shaker until the SDS was removed completely. After

washing in the triton X-100, the gel was incubated in the 50 mM phosphate buffer

(pH -7) at 37 "C for 4 hr. After incubation, the gel was stained with CBBR-250 (as in

SDS-PA.GE). The over stained gel was destained in low destaining solution so that

white bands of proteases could be visible in dark background. The zymogram

containing various white protease bands was scarmed immediately and the data was

saved for further analysis.

Staining procedure for GST in native gel:

To detect GST activity in a native PAGE gel the method of Ricci et al, (1984)

was followed. The electrophoresed gel was placed in 0.1 M potassium phosphate

buffer (pH- 6.5), containing 4.5 mM GSH, 1.0 mM CDNB, and 1.0 mM NET.

Incubations were performed in a metabolic shaker at 37°C with gentle agitation. After

10 min, gels were washed with water and incubated in 4.0 ml solution of 0.1 M Tris-

HCI buffer (pH- 9.6), containing 3.0 mM PMS at room temperature. Blue insoluble

formazan appeared on the gel surface in about 3-5 min of incubation except in the

GST area. The gel was scanned immediately and the data was saved for further

analysis.

23

Molecular Weight Determination: -The molecular weights of protem bands

obtained after SDS-PACiK, native PAGE, and gelatin substrate z>mograph> were

determined by the following method.

The relative mobility (Rj) of each polypeptide was calculated as follows*

Distance of migrated polypeptide Rf Distance of migrated tracking dve

The Rf value of individual polypeptide was calculated with the help of their

migration distance. Subsequently molecular weight (Mr) of each polypeptide was

determined with the help of a standard graph of known molecular weight marker

(PAGE mark ^^ MID prestained marker with molecular weight range of 19.2-108

kDa, G-Biosciences, St Louis, MO, USA) proteins against their Rf values.

rSOELECTRIC FOCUSSING (lEF):

The lEF was performed following the method of O' Parrel (1975) for

separation of polypeptides based on their isoelectric point (pi). The lEF was

performed using LKB 2117 Multiphore with 2197 power supply (Pharmacia, LKB).

Preparation of gel (10 ml):

Acrylamide/bisacrylamide 1.67ml (28.38% / bisacrylamide 1.6%)

Urea 4.8 gm (8M)

CHAPS (10%) 2.0 ml

Ampholyte 0.25ml

(pH,3-10)

Dissolve and maintain volume to 10 ml with distilled water.

APS (10%) 60^1

TEMED 6 nl

Sample buffer: : The sample buffer consists of 7 M urea, 2 M thiourea, 4% (w/v) Chaps, 66 mM DTT and 0.5% (v/v) pharmalyte (pH 3-10). Sample was mixed in sample buffer in 1:1 ratio.

24

Gel casting and electrofocussing: The gel solution was poured in between the lEF

glass plates, separated by a 1 mm thick spacer and allowed to polymerize at room

temperature for 1 hour.

After polymerization, the gel was transfeiTed to thin glass plate. The glass plate

alongwith the gel was transferred to cooloing plate of the Multiphore assembly which

was maintained at 8-10 ''C by Multitemp II thermostatic circulator. At cathode

position, an electrode wick soaked in 1 M NaOH solution was placed while at anode

position, the electrode wick soaked in 1 M H3PO4 solution was placed. The lEF gel

was prefocussed at 100,150 and 200 volts for 15, 30 and 30 minutes respectively.

After prefocussing, samples containing approximately 100 |.ig proteins were placed on

to the gel using sample applicator strip and focussed at 20 watt (constant) for 45

minutes. Thereafter, the sample strips were removed and again focussed at 25 watt

(constant) for another 3 hrs. After the completion of electrofocussing, the electrode

wicks were removed and the gel was immediately fixed in the fixative solution

containing 3.46% (w/v) sulphosalicyclic acid and 11.5% (w/v) trichloroacetic acid in

double distilled water (DDW). After fixation, the gel was washed with destaining

solution which contains 25% v/v methanol and S% v/v acetic acid in DDW. The gel

was then stained with CBBR-250 as mentioned earlier. Gel was destained until a clear

back gorund with distinct polypeptide bands were visible. Gel was scanned and data

stored for further analysis.

Spectrophotometric analysis of enzj mes:

Enzyme activity of acid phosphatase, alkaline phosphatase, glutathione S-

transferase, GOT and GPT was determined by standardized spectrophotometric

methods on UV-1700 pharma spectrophotometer (Shimadzu, USA).

1. Acid Phosphatase Assay (E.C. 3.1.3.2):

Enzyme activity of acid phosphatase was determined following the method as

described in Bergmeyer et al. (1974). The assay mixture with a total volume of 1.1 ml

contained 0.28 ml of 0.05 M acetate buffer (pH 5.0), 8 mM substrate (p- nitrophenyl

phosphate, Sigma chemical Co, U.S.A.) and 0.02 ml protein sample. The enzyme

reaction was allowed for 60 minutes at 37 °C and thereafter stopped by adding 0.02 N

25

NaOH. The liberated p-nitrophenvl was measured at 420 nm. Finally, the enzyme

activit) was calculated with reference to a previoush calibrated curve of known

concentration of p-nitrophenyi (Sigma chemical Co, U.S.A.)

2. Alkaline Phosphatase Assay (E.C.3,1.3-1):

Alkaline phosphatase activity was determined following the method as

described in Bergmyer et al, (1974). The assay mixture contained a loial volume of

1.2 ml having 1 ml of 0.05 M glycine NaOH buffer (pH 9.5), 5 mM enzyme substrate

(p-nitrophenyl phosphate) and 0.02 ml of enzyme preparation. For enzyme reaction,

the sample was incubated for 1 hr at 37 C. After incubation the enzyme reaction with

substrate was stopped by adding 0.02 M NaOH. The liberated p-nitrophenyl was

measured at 420 nm. The enzyme activity was calculated as mentioned for acid

phosphatase.

3. Glutathione- S transferase (GST) Assay (EC 2.5.1.18):

GST activity was assayed following the method of Habig et al, (1974). The

assay mixture with a total volume of 3 ml having 2.64 ml 0.1 M phosphate buffer, 0,3

ml of 0.1 mM reduced glutathione, 0.0! m.I CDNB (l-chloro-2.4-dinitrobenzene) and

0.05 ml sample containing enzyme. A complete assay mixture without enzyme was

used as a control. Changes in optical density were measured after every 30 seconds

upto 1 minute in a spectrophotometer at 304 nm.

4. Glutamate Oxaloacetate Transaminase Assay (GOT) (E.G. 2.6.1.1):

For GOT enzyme assay an enzyme kit from Span diagnostic company (Surat,

India) was purchased and the company instructions were followed. The enzyme assay

principle is based on Reitman and Frankel Method (1957).

Assay Principle:

Aspartate aminotransferase (AST) is known to catalyse the transamination of

L-Aspartate and a- Ketoglutarate (a-KG) to form oxaloacetate and L-Glutamate. The

oxaloacetate formed reacted with 2-4, Dinitrophenyl hydrazine (2-4 DNPH) to form a

corresponding hydrazone, a brown coloured complex in alkaline medium which was

measured spectrophotometrically at 505 nm.

Reactions are as follows:

1 a- Ketoglutai"ate + L- Aspaitate ^^"^=~^ Oxaloacetate + L- Glutamate

2. Ocaloacetate + 2,4-DNPH : : ; := :" Corresponding Hydrazone (browTi colour)

5. Glutamate Pyruvate Transaminase Assay (GPT) (E.C 2.6.1.2):

GPT enzyme assay was performed using Span diagnostic kit (Span Diagnostic

Ltd Surat India). The assay principle is based on Reitman and Franlcel Metliod (1957)

wliicli iias been used for the estimation of GPT enzyme activity for the present study.

Assay Principle:

Alanine aminotransferase (ALT) enzyme is known to catalyse the

transamination of L-AIanine and a- Ketoglutarate (a-KG) to form pyruvate and L-

Glutamate. The pyruvate then reacts with 2-4, Dinitrophenyl hydrazine (2, 4 - DNPH)

to form a coaesponding hydrazone, a brown coloured complex in alkaline medium

and this brown colour has been measured spectrophotometrically at 505 nm.

Reactions are as follows:

1. a- Ketoglutarate + L- Aspartate ^ Oxaloacetate + L- Glutamate

2. Oxaloacetate + 2,4-DNPH -, ^ Corresponding Hydrazone (browTi colour)

27

m

RESULTS

m

Results

During the present investigation a number of biochemical studies like analysis

ot total protein, enz}me turn over, polypeptide profile, protease profile and GST

enzyme profile ha\c been performed The study includes adult and miracidial

developmental stages of amphistome parasites infecting liver and rumen of Indian

water buffaloes. The larval stages of Gastrothylax crumenifer (rumen) and

Gigantocotyle explanatum (liver) include zero day (fresh) eggs, 4-day embryo and

mature miracidia.

The data have been analysed and a comparison has been made among the

miracidial developmental stages of the amphistomes infecting two different habitats.

Results are summarized in Figures, 4-15 and Tables, 1-6.

Some enzymes like acid phosphatase (AcPase), alkaline phosphatase

(AlkPase), glutamate-oxaloacetate transaminase (GOT), glutamate pyruvate

transaminase (GPT) and glutathione S- transferase have been studied in adult and

miracidial developmental stages of both the parasites. Marked differences have been

observed in the enzyme turn over during the development (Figures, 4-9 Tables, 2 and

3). Likewise variations in the total protein content (Figures, 1 and 2 and Table, 1) in

the two amphistome species have also been observed during the present study.

Expression of polypeptides and isozymes has been studied using SDS-PAGE,

lEF, native PAGE and gelatin substrate zymography, (Figures, 10, 11, 12, 13, 14 and

15 and Tables, 4, 5 and 6).

1- Level of protein during tlie miracidial development:

Differences in the protein concentration of adult, 0-day eggs, 4-day eggs and eggs

containing mature miracidia (Em) of both Gastrothylax crumenifer and Gigantocotyle

explanatum have been observed. A steady decline in the protein content from freshly

collected eggs to the eggs containing mature miracidia (Em) was observed. Highest

protein content has been recorded in the adults. The protein content decreased by 1.20

folds from 0-day eggs to 4-day eggs and by 1.69 fold from 4-day eggs to Em stage

in the case of G. crumenifer. Where as in the G. explanatum the protein content

was decreased by 1.24 fold from 0-day eggs to 4- day eggs and by 1.44 fold from 4-

28

day eggs to Em. Analysis of protein content at interspecific level also revealed clear

differences. It was observed that in rumen amphistomes as compared to liver

amphistomes the level of protein was more during miracidial and in the adult stages

The data revealed 1.42, 1.46 and 1.24 fold higher proteins in 0-day eggs, 4-day eggs

and Em eggs of O'. crumemfer respectuely as compared to G. explanatum, (Table, 1).

Further, the adult G. crumenifer revealed 1.29 fold higher proteins as compared to

adult G. explanatum (Table, 1 and Figures, 4 and 5).

2- Acid phosphatase:

On comparing the acid phosphatase enzyme activity among 0-day, 4-day eggs

and Em it was observed that the enzyme activity followed an increasing trend from

one developmental stage to another in both the amphistome species under study. In G.

crumenifer, the enzyme activity increased by 1.131, 1.341 and 1.120 fold from 0-day

eggs to 4-day eggs, 4-day eggs to Em and Em to adult stage respectively. Similarly,

increased in enzyme activity by 1.051, 1.302 and 1.252 fold from 0-day to 4-day eggs,

4-day eggs to Em and Em to adult stage respectively was recorded in G. explanatum

(Tables, 2 and 3; Figures, 6 and 7).

3-AIkaline Phosphatase:

Analysis of alkaline phosphatase activity during the miracidial development of

rumen and liver infecting amphistome parasites revealed a similar pattern as recorded

for acid phosphatase enzyme. Elevation in the enzyme activity was noticed in the

successive developmental stages that is from 0-day eggs to 4-day eggs, mature

miracidia and adult stages. Alkaline phosphatase enzyme activity revealed 1.052,

1.635 and 1.46 fold elevation from 0-day eggs to 4-day eggs, 4-day eggs to mature

miracidia (Em) and Em to adult stages in the rumen parasite G. crumenifer while

elevation in enzyme activity by 1.089, 1.647 and 1.113 fold was recorded in the

G.explanatum (Tables, 2 and 3; Figures, 6 and 7) respectively.

29

4- Glutathione-S-transferase (GST):

Glutathione-S-transferasc enzyme activil} was recorded in the adult as well as in the

miracidial de\'elopnienta] stages of both G crumenifer and G. explanatnm during the

present stud}.

Enzyme turn over in adult stages of both the parasites was considerably higher

as compared to larval developmental stages. In G. crumenifer, 0-day to 4-dayeggs, 4-

day eggs to Em and Em toadult stages revealed 9.30, 53.81 and 167.57 percent

mcrease while 21.7, 60.91 and 104.91 percent increase in enzyme activity was

observed in G. explanatum (Table, 2 and 3, Figures, 8 and 9) respectively .

5- Glutaraate oxaloacetate transaminase (GOT):

Analysis of enzyme assay data revealed elevation in the GOT enzyme activity

from one developmental stage to the next larval stage. In G crumenifer it was noticed

that from 0-day eggs to 4-day eggs GOT level increased by 37.5 percent while from

4-day egg stage to Em and Em to adult stage enzyme activity increased by 30.24 and

65.96 percent respctively. In G. explanatum, a similar trend in the elevation of

enzyme activity from zero to 4- day eggs (42.52 percent) 4-day eggs to Em (32.54

percent increase) and Em to adult (50.76 percent increase) stage was observed

(Tables 2 and 3 and Figures Sand 9).

6- Glutamate pyruvate transaminase (GPT):

Comparision of GPT enzyme activity in different developmental stages of

miracidia revealed higher enzyme activity from zero day eggs to 4-day eggs, 4-day

eggs to Em and Em to aduh stage by 1.318, 1.947 and 1.505 fold respectively in G.

crumenifer. A similar trend was noticed in the developmental stages of G.

explanatum. The data revealedl.32, 2.288 and 1.344 fold increase from 0-day eggs to

4-day eggs, 4-day eggs to Em and Em to adult stage respectively (Tables, 2 and 3 and

Figures, 8 and 9).

30

Table 1: The level of protein content in 0-day eggs. 4-day eggs, eggs containing

mature miracidia (Em) and adults of GastrGthylax crumenifer and Gigantocotyle

explanatum.

1

Stages

Adult

0-day eggs

4-day eggs

Em

Gastrothylax crumenifer

(mg/gm wet weight)

69.033±2.99

33.33±2.306

27.56±1.256

16.23±0.560

Gigantocotyle explanatum

(mg/gm wet weight)

53.50 ±2.971

23.460 ±L198

18.800 ±0.687

13.033 ±0.375

All the values are mean of three replicates.

±SEM.

All the values are calculated at p< 0.05 significance level.

31

Gigantocotyle explanatum

£

10

4.

Adult 0 d.is 4 rlov Cm

Figure 4: The level of protein in O-day eggs, 4-day eggs, eggs containing mature

miracidia f Em) and adult of Gigantocotyle explanatum. Error bars are showing ±

SEM

• s f i

/o

' ,0

t 0 s 4 - '

Qfl 50

.^0

iO

Gastrothylax crumenifer

I Aduil 0 dciy 4 dav f n^

Figure 5 The level of prote in in O-day eggs, 4-clav eggs, eggs containing mature

miracidia (Em) and adult of Gastrothylax crumenifer Error bars are showing ±

SEM

Table 2: Shows the levels of different enzyme activities in 0-day eggs, 4-day eggs,

eggs containing mature miracidia (Hm) and adults of Gaslrolhylax crumenifer.

Enzymes

*Acid

phosphatase

^Alkaline

phosphatase

-i^GST

*GOT

^GPT

Gastrothylax

crumenifer

(Adult)

5.251±0.139

9.184±0.155

6.692±0.132

L185±0.059

A OO/C 1 A OTA

0 day eggs

3.058±0.0342

4.654±0.100

1.667±0.127

0.416±0.014

A O i / i r m m O . Z l t : n . u i U l

4 day eggs

3.495± 0.172

4.900±0.057

2.029±0.194

0.593±0.05

U.ZOZaiU.UZJ

Em

4.688±0.237

8.010±0.137

3.265±0.120

0.786±0.034

0.549±.0184

*Enzyme unit: |ig of pnp liberated /mg of protein /minute

•ii'Enzyme unit: lU/ mg of protein/minute

All the values are mean of three replicates.

±SEM.

All the values are calculated at p< 0.05 significance level.

34

lable 3: Shuv\h the lexei. of clifteient en/vnie acti\ities in !)-da\ eggs. 4-da} eggs,

eggs containing mature miracidia (Em) and adults of Gigimtocotyh' explanatum

! Enzvme Gigantocotyle \ 0-da> eg&s i 4-da^ eggs T Em

i

} "Acid phosphatase 1

"Alkaline

phosphatase

^ S T

^ O T

^ P T

explanatum

(adult)

5.863±0.077

8.841±0.093

8.458±0.254

1.19±0.065

0.766±0.02I

3.418±0.063

4.422i=07078 ""

1.88±0.054

0.401±0.026

0.202±0 023

3.594±0.090

4.819±0.031

2.055±0.099

0.55±0.039

0.249±0.0!5

4.681±0.150

7.94±0.044

0.717±0.031

0.570±0.021

* Enzyme unit: )ig of pnp liberated /mg of protein /minute

•5 Enzyme unit: lU/ mg of protein/minute

All the values are mean of three replicates.

iSEM.

All the values are calculated at p< 0.05 significance level.

35

1 0 Gastrothylax crumenifer

I '

E

^

•S 3

Adult O-day 4-dav Em Figure 6- The levels of acid and allcaline phosphatase activit ies in 0-day, 4-day, Eggs

containing mature miracidia (Em) and adult of Gastrothylax crumenifer. Error bars are showing ± SEM.

36

10

8

I, *5 6 o

2 3 «

a. c

o

Gigantocotyle explanatum

i i AcPase w AlkPase

Adult 0-day 4-dav Em

Figure 7: The level of Add and alkaline phosphatase in O-day eggs, 4-day eggs, eggs containing mature miracidia (Em) and adult of Gigantocotyle explanatum. Error bars are

showing ± SEM.

37

Gastrothylax crumenifer

I GST • GOT u GPT

5 5

I. ^ 3

Adult 0-day 4-day Em Figur* 8: Th« Uval of activity of GST, GOT and GPT of 0-day aggt, 4-day aggs, aggs

contaiaing mature miracidia (Em} and adult of Gastrothylax crumenifer. Error bars arc showing tSEM.

38

10

9

8

i:

I' 2 3

2

1

0

Gigantocotyle explanatum

1 ILJLJJ^

Adult 0-dav 4-daY Em

Figure 9: The level of GST, GOT and GPT in 0-day eggs, 4-day eggs, eggs containing mature miracida |Em) and adult of Gigantocotyle explanatum. Error bars are showing ± SEM.

39

SDS-PAGE POLYPEPTIDE PROFILE:

In the present study SI3S PAGt was performed to investigate the polypeptide

profile during tlie miracidial development. The electrophorcsed gels were stained with

coomassie brilliant blue R-250 as well as with silver stain. More protein bands were

visible b> silver staining as compared to coomassie staming; therefore silverstained

gel was used for polypeptide profile analysis. The analysis of resolved polypeptides

revealed that the individual proteins of each stage of eggs separated into

heterogeneous mixture of polypepfides of varying molecular weights. A total of 30,

31- 33 and 34 protein bands in zero day eggs, 4 day eggs, Em and adult stages

respectively, were present in G. explanatum wherease 22, 25, 26 and 34 protein bands

resolved in 0-day eggs, 4-day eggs, Em and adult stages of G. crumenifer

respectively. Results are shown in Table, 4 and Figures, 10 and 11.

The protein profile revealed quantitative and qualitative differences besides

some fundamental similarities during the development from one stage to another

under investigation. On the basis of present data the polypeptides can be grouped into

three categories - conserved polypeptides, stage-specific polypeptides and variable

polypeptides

1-Conserved polypeptides: Polypeptides which were present in all the miracidial

developmental stages (0-day, 4-day, Em) and adult stages of G.explanatum and G.

crumenifer were considered as conserved polypeptides during the present study.

2-Specific polypeptides: Those polypeptides which were present only in a particular

developmental stage under study were categorized as specific polypeptides.

3- Variable polypeptides: The polypeptides which were inconsistent in their

presence during the course of development under investigation were considered as

variable polypeptides.

Conserved polypeptides: During the present study, the polypeptides of

molecular weight 150, 140, 104.5, 102, 48, 37, 33 20.5, and 19.5 kDa were considered

as Conserved polypeptides because of their presence in all the developmental stages

of G. explanatum whereas polypeptides of 150, 57, 46, 40, 35, 22.5, 19.5, and 19 kDa

40

were consideicd as conserved which are present m all the developmental stages of G

Specific polypeptides: There were 6, 8, 9 and 14 specific protein bands in 0-

dav eggs, 4-day eggs. Em and adult of G. explanatum having molecular weights of

121.5, 84.5, 57, 47, 39.5, and 24.5 kDa in zero day eggs while 125, 114 , 90 , 67, 60,

40, 31.5 , 28.5, and 20 kDa in 4-day eggs, 124.5, 116, 86.5, 74, 72.5 , 50 ,48.5, 44,

28 kDa in Em and 151, 130, 125, 123, 122, 118, 116, 78, 49, 43, 41, 39, 34, 29.5 kDa

in adult stages. A total of 5, 6, 7 and 13 specific protein bands were found in 0-day

eggs, 4-day eggs, Em and adult of G crumenifer respectively, with molecular weight

of 104, 58. 50, 38 and 28.5 kDa in 0-day eggs, 66, 54, 45, 32, 29, and 27 kDa in 4-day

eggs 118, 108, 101.5, 76, 61,27.5, and 21 kDa in Em and 148, 133, 125, 107,94,81,

78, 70, 65, 48. 33, 28 and 26.5 kDa m adult stages.

Variable polypeptides: The polypeptides of apparent moleculr weight of 131,

120,108, 94, 86.6, 84.5 70, 54, 42, 38, 31, 29, 27, 25, 23, 21 kDa were considered as

variable polypeptides in case of G explanatum where as polypeptides of molecular

weight 135, 129, 90, 114, 112, 69, 62, 43, 34, 31, 25.5 kDa were considered as

variable polypeptides incase of G crumenifer because of their inconsistent

appearance in all the developmental stages.

41

-a

c

cd

«

afi

o o

00 u >-> as

CO aj

-o 4 - *

H) a , >. o t.2,

O

<u

o

w O < D H

C/0

Q 0 0

-a

c rt en

U >

00

T t

a> X> a H

S •>j

t - .

C3 C M

~ i :

i j

« _bc O

c3 5^

s* <iJ

S a ^ o

J >> •i£

Ol CS O

s is 5

^ ^

•-S -&» © o C5 •*« s: 55 .2f 6

&. ^ ••«> s:

s b y

"CJ

<«« 5i i '•"'A CO

3

T~

t*-. O

^

s

o u

E s a "a o H

o

6 s

M H «

O

H

O

u :^ ? i

S

s

o H

t M O

6

o H

6«

2

u

w C !/:

u

s *3 c 1

C3

2 -^ C^ o CM

"c c.

w SS

(U

O

W3 4^

•o * ^ 4

o c

2 a. a

"c.

2 *-*-* a

« M 4

CU en

2

a.

c

r i

o ' r^l

, , i r^

^

=3-m

ir-T

r* n

oc'

^

m

^ m

• * j

3

- t CO

T — -

iXj'

. ,

oo

-*' ON

o

i n

O-' r - i

^ - » -

i n °« .A ' * ^ ^Nl

^ o6

1 -

, O iy^ r o

r j

' '

• o

o m

i n

^ • o "^ «m 0,^

' '

i n

CN ( N

Vl M feC u ^

T3 1

O

o

1 — 1

m . ( N

1-

OO

r—

m

tnT ^ .'

i n

^ VO

IZl ftXl bc

•a 1

o

<n' oc

i n

m

t~~ CN

^.^ <N

\ o

^o <N

i n

oo

oc

'—' • r f ( N •r—H

ON

m CO

t ^ ^ oo

^ od"

r--

0 0 ( N

s

00 (N

oo

o ' i n

m ( N r~

OJ

i n "

c3

3 o

•108

"78.6

"50.6

•35.9

•27.1

•19.2

Marker

Figure 10: Coomassie brilliant blue (R-250) stained polypeptide profile of 0- day

eggs, 4- day egg and eggs containing mature miracidia (Em) and adults of

Gastrothylax crumenifer and Gigantocotyle explanatum.

43

108

'78.6

>50.6

•35.9

•27.1

'19.2

G. crumenifer 6. explanatum

Figure 11: Silver stained polypeptide profile of 0-day eggs, 4-day eggs, eggs

containing mature miracidia (Em) and adults of Gastrothylax crumenifer and

Gigantocotyle explanatum.

44

Double staining: In addition to CBBR'250 and silver staining of individual

clcctrophorcsbed gel containing polypeptides of miracidial developmental stages of G

crumenifer and G. explanatum, a double staining procedure was also performed.

Using double staining meuiod, three categories of polypeptides with blue, yellow or

brown colours was recorded in the present study. It was observed that 11. 9 and 8

protein bands with blue stain resoh'ed in 0-day eggs, 4-day eggs and eggs containing

mature miracidia (Em) respectively, with a m.olecular weight range from 25 to78 kDa

in the G. explanatum whereas miracidial developmental stages of G. cromenifer did

not show blue colour and all the bands were either of yellow or brown colour (Figure.

12). All the yellow and brown bands were in the molecular weight range of 19-151

kDa in case of both.

Isoelectric focusing (lEF):

In the present study, proteins obtained from adult G. crumenifer and G. explanatum

were subjected to lEF. The lEF profile (Figure, 13) was analysed and it was observed

that most of the polypeptides focused in the acidic range under study. As shown in

Figure 13, in G. explanatum 6 polypeptides were focused in the acidic range while 4

polypeptides were focused in the alkaline range. Similarly, in G. crumenifer, 5 and 4

polypeptides were focused in the acidic and alakaiine range respectively (Figure, 13).

45

4 i^Y E'-

G. explanatum G. crumenifer

Figure 12: Showing dcnible stained poh peptide profile of 0- da\ eggs. 4-da\ eggs

and eggs containing mature miracidia of G explanatum and G crumenifer.

Figure 13: Isoelectric focusing gel showing pi of adult d explanatum and (/

crumenifer

46

Gelatin Substrate Zymography:

In order to investigate protease profile of O-daj' eggs. 4-day eggs, Em and

adult stages of G. explanatum and G. crumeifer. gelatin substiate zymography was

performed A zymogram was developed and analysis of the protease profile revealed

inconsistency in their presence in the developmental stages of both the parasites under

study. No protease was obtained in the 0-day eggs of Gigantocotyle explanatum. A

total 5, 6 and 2 protease bands were resolved in 4-day eggs, Em and adult stages.

Proteases with molecular weight of 123, 102.5, 92, 86, and 53 kDa were resolved in

4-day eggs while 123, 120, 102.5, 92. 80. and 53 kDa proteases in Em and 123, 35.9

kDa proteases were resolved in adult of G. explanatum. In G.crumenifer a total of 1,

5, 7 and 2 bands of proteases were resolved in 0-day eggs, 4-day eggs, Em and adult

respectively, with apparent molecular weight of 123 kDa in zero day eggs, 123, 120,

105, 50, 30 kDa in 4-day eggs, 123, 108, 105, 50, 37, 34 and 30 kDa in Em and 123,

78.6 kDa in adult stages respectively. Results are sshown in Table 5 and Figure 14.

Like polypeptide profile, protease bands also show quantitative and

qualitative differences in their profile during the present investigation. Proteases

resolved in to conserved, variable and specific polypeptides.

Conserved polypeptides: Proteases of molecular weight 123 kDa is considered to be

conserved during the miracidial development of both the parasites.

Variable: A total of 3 protease bands were considered to be variable with molecular

weight of 102.5, 92, and 53 kDa in 0-day, 4-day and Em of G. explanatum

respectively. In case of 0-day eggs, 4-day eggs and Em of G. crumenifer three

protease bands were considered to be variable having molecular weight of 105, 50 and

30 kDa respectively.

Specific proteases: In 0-day eggs, 4-day eggs, and adult of G.explanatum, a total of

1, 2, and 1 protease bands are found to be specific with a molecular weight of 86 kDa

in 4 day eggs, 120, and 80 KDa in Em and 35.9 kDa in adult stages respectively,

whereas a total of 1 (120 kDa), 3 (108, 37, 34 kDa) and I (78.6 kDa) protease bands

were found to be specific for 4-day eggs, Em and adult stages of G. crumenifer

respectively.

47

Table 5: Profile of proteases in 0-day eggs, 4-day eggs and eggs containing mature

niiracidia (Em) of Gastroihylax crumenifer and Gigantocotyle explanatum.

Gastrothylax cnimen ifer

Stages

Adult

0-day eggs

4-day eggs

Eggs

containing

mature

niiracidia

VIW-indicate5

Total no.

of

proteases

2

1

5

7

> molecular \

Total

number

of stage-

specific

proteases

1

0

1

3

veight

MW of

specific

protease

(kDa)

78

-

120

108, 37,

34

Gieantocotvie

explanatum

Total no.

of

proteases

2

0

5

6

Total

number

of stage-

specific

proteases

1

0

1

2

MW of

specific

proreases

(kDa)

35.9

-

86

120, 80.

48

108 kDa

78.6 kDa

G. explanatum

Figure 14: Zymograme of proteases of 0- day eggs, 4- day eggs. Eggs containing

mature miracidia (Em) and adults of Gastrothylax crumenifer and Gigantocotyle

explanatum.

49

Isozyme profile of GST: GST Isozymes in 0-day, 4-day, Em and adult of G.

crumenifer and G. explanatum were resolved by native PAGE. A total of 3, 3, 5 and 5

isozymes of GST were observed in 0-day eggs, 4-day eggs, Em and adult of G.

crumenifer respectively. In G. explanatum a total of 3, 4, 6 and 6 isozymes of GST

were observed in 0-day eggs, 4-day eggs, Em and adult stages respectively. Results

are shown in Table 6 and Figure 15.

Specific isozymes: A total of 2 and 4 isozymes of GST with a molecular weight of 19

andl7.5 kDa in Em and 118,104, 52 and 41 kDa in adult of G. crumenifer were found

as specific, whereas no specific isozyme was observed in 0-day eggs and 4-day eggs.

A total 1, 2 and 2 specific isozymes resolved in 4-day eggs, Em and adult of G.

explanatum respectively. The apparent molecular weight of 78 kDa in 4-day eggs,

22.5, and 19 kDa in Em and 91, 25 kDa in adult of G. explanatum were evident. No

specific isozymes were observed in 0-day eggs of G. explnatum.

Conserved isozymes: Only one GST isozyme was observed as conserved with a

molecular weight of 120 in all the developmental stages of G. crumenifer. While in

case of G. explanatum three isozymes of GST were considered as conserved having

moleculae weights of 120, 67 and 49 Da.

Variable isozymes: Two bands of GST isozymes wereobserved with molecular

weight of 39 kDa and 34. 5 kDa in G. crumenifer while in G. explanatum only one

variable isozyme appeared with a molecular weight of 20 kDa.

50

Table 6: Isozyme profile of Glutathione- S transferse (GST) in 0-day eggs, 4-day

eggs, eggs containing mature miracidia (Em) and adults of Gastrothylax crumenifer

and Gigantocotyle explanatum.

Stages

Adult

0-day

eggs

4-day

eggs

Em

Gastrothylax crumenifer

Total no.

of

isozymes

5

3

3

5

Total

number

of stage-

specific

isozymes

4

0

0

2

MW of

specific

isozymes

(kDa)

118, 104,

52,41.

19,17.5.

explanatum

Total no.

of

isozymes

6

3

4

6

Gigantocotyle

Total

number

of stage-

specific

isozymes

2

0

1

2

MW of

specific

isozymes

(kDa)

91,25.

78

22.5,19.

MW-indicates molecular weight

51

108

78.6

50.6

• 35.9

27.1

19.2

Marker

Y' G. crumenifer

Figure 15: Enzyme profile of Glutathione S-transferase of 0-day eggs, 4-day eggs,

Eggs containing mature miracidia (Em) and adults of Gastrothylax crumenifer and

Gigantocotyle explanatum.

52

m m

m •pji

Discussion

The present stud) was focussed on to characterize stage- specific

pohpeptides during the miracidial development of Gaslrothylax crumenifer and

Giguntocotyle explanalum because the proteins are the integral component of every

biological acti\'ily: thc> pla> a significant role in physiology and are distributed

ubiquitously. Beside their primary role they are also involved in the contractile

system, in transport mechanism, as protective agents, act as hormones, serve as amino

acid reserves and also act as a source of energy in the absence of primary

(carbohydrate) energy source. During cellular differentiation the nucleocytoplasmic

interactions lead to the qualitative and quantitative biochemical changes that

ultimately lead to morphogenesis. The proteins are of obvious interest as the

development of new structures and physiological activities are linked with these

molecules including the enormous fecundity.

The result of the present study reveals that protein reserves of the parasites

change at different stages during the development of miracidia. The gradual decline in

protein content of the eggs during the miracidial development in the present study

suggest that proteins may be involved in cellular differentiation and organogenesis,

energy production and other metabolic processes which are activated during

development. The decline in the level of proteins of the developing embryo of G.

explanatum and G. crumenifer may be intrinsically programmed for the utilization of

food. However, the keratin type egg shell protein of G. explanatum and G. crumenifer

(Arfm and Nizami, 1986) may remain constant and the tissue protein being utilized

during the developmental process. In a similar study, Wilson (1967) also reported a

significant decline in the protein content and carbohydrate reserves of the egg during

the development of F. hepatica. The protein synthesized under the control of maternal

genes which are gradually replaced during the development by the protein synthesized

under the control of embryo genes (Khrushchov, 1981). The metabolic turn over

accompanying the transition from one stage to the next involves qualitative as well as

quantitative changes in various components which may be greatly influenced by

various physico-chemical factors of different micro-habitats. The wide variation in the

protein content of different parasites belonging to the same or different taxonomic

group and inliabiting various micro- environments indicate that no generalization is

53

possible. In the prese?it study it is evident that habitat influences the protein content

and d considerable quantitative \driation m protein content vs-as noticed when the

results of (T. cnimenifer collected from the rumen of buffalo and G explanatum

collected from the Ii\er of buffalo were compared.

The present stud\ was carried out based on the hypothesis that the stage-

specific polypeptides are associated with differentiation and organogenesis of the

parasite. The 0-day eggs, 4-day eggs, eggs containing mature miracidia (Em) and

adult stages of the Gigantocotyle explanatum and Gastrothylax crumenifer were used

to perform the current study. The protein profile was analyzed by SDS-PAGE.

Among various gel staining techniques, Coomassie brilliant blue R-250 (CBBR-250)

is the most commonly used technique for visualizing polypeptides in polyacrylamide

gels. But, due to comparatively low sensitivity of CBBR-250 staining over silver

staining, most laboratories use the silver staining technique which is 100 fold more

sensitive than CBBR-250 staining for detecting polypeptides on polyacrylamide gels

(Switzer et al., 1979, Oakley et al, 1980).

The number of demonstrable polypeptides in a gel depends upon the

sensitivity of staining technique to different polypeptides, not all proteins stain

equally with coomassie brilliant blue, highly glycosylated proteins like glycophorin

stain poorly with coom-assie dye. Simdlarly, Switzer et al, (1979) and Oakley et al,

(1980) have also pointed out limitations of coomassie brilliant blue staining and

insensitivity for conjugated proteins. In the present study the number of polypeptides

which are detected by silver staining is much higher as compared to the number of

polypeptides stained by coomasie staining. The difference in the polypeptides may be

due to differential affinity of two staining techniques towards different conjugated

proteins. Proteins which are present in traces and those heavily glycosylated as well as

lipoproteins could not be detected by coomassie brilliant blue staining (Sammons et

al, 1981).

A complex protein profile was obtained by CBBR-250 and silver staining

procedures and the complexity increases with the maturation of the miracidium and

adult. The Silver staining technique revealed comparatively large number of

polypeptides with apparent molecular weight range of 19 to 151 kDa. Moreover

majority of polypeptides which are visualized by coomassie brilliant blue, were

detected by silver staining. Due to the complexity of polypeptide, they were grouped

54

.>i' ' 'i -

into tlirec categories (Conserved, stage-specific and \'ariablc) acrofding iD-lheir'

presence and absence durnig development Such complexities are expected for a

metazoan parasite, which possess various tissue and organ systems, appear at different

phases of development. Polypeptides having molecular weight of 150, 140, 104.5,

102, 48, 37. 33. 20 5 and 19.5 in Gigantocolyle explamtum and of 150. 57. 46, 40, 35,

22.5, 19.5 and 19 kDa in Gastrothylax crumenijer were detected in all the

de\'elo'^mental sta< es and referred as conserved '^oly'^e"tides. These are "resumabl^'

basic component of structural and functional organization of the parasite and therefore

being continuously synthesized throughout the development of the parasite. Saifullah

(1999) studied stage-specific polypeptides in 0-day eggs, eggs containing mature

miracidia (Em), cercariae and metacercariae of Gastrothylax crumenifer. However in

F hepatica, when soluble proteins of 4.5, 8, 11 and 18 old week fluke were analysed

by SDS-PAGE. Majority of poypeptides (Having molecular weight of 220, 200, 170,

160, 150, 136, 125, 100, 82, 73. 65, 58, 57, 56, 50, 35, 33, 29, 25, 23 kDa) were found

conserved during the development (Anderson, 1989).

The surface proteins of schistosomula and adult S. mansoni, revealed that

majority of polypeptide with a molecular weight of 150, 105, 100, 85, 78, 68, 55, 46,

40, 36, 26.20 and 16 kDa) were observed in both stages. However, polypeptides ^•^.^^'.f.^ f^~ „^u;„+.,„«..«,,1« «„J „ j , . u „ „,„„„ „i„„ ; j „ „ * : f ; „ j /nun:— i T-> : u apc»./in\.- lu i 3»^iiisnjsuiiiula aiiu auu i ia w c i c a i su luc i i i i i i cu (^rimip oiiu ivaiiijcincj^is.,

1984). The stage-specific polypeptides for each developmental stage may have strong

co-relation with the morphological development. Therefore the characteristic

polypeptides detected in these stages which could be required for a particular fimction

at particular stage of development or transitory proteins which may be utilized during

subsequent stages of development. A number of stage-specific isozymes have been

demonstrated in the developmental stages of H. diminuta (Wolkey and Fairbaim

1973). The third group is of variable polypeptides which showed inconsistency

during the development. Appearance of polypeptides coincides with the development

of reproductive organs of the parasite, indicating that these proteins are constituent of

the reproductive organ. However, further studies are required to ascertain their

function and biochemical nature.

These three categories of polypeptides substantiate the hypothetical model

proposed by Rogers and Petronijevic (1982) for the control of development in

nematodes. Authors suggested that development is controlled by a set of genes so that

55

each iife cycle siage iias d cuiicspunding ^et uf gene. Some of thche genes are stage-

specific other may be active at more than one stage of Hte cycle, although their

control mechanism may change The gene sets are switched on and off at appropriate

point of the life cycle, although this may involve sequential rather than block changes.

The threshold for switching can var> throughout life cycle.

On the basis of above discussions it can be concluded that the identified

polypeptides showed definite relationship Vv'ith physiological and miorphological

development of parasite.

By double staining the blue bands obtained can be considered as basic proteins There

are more basic proteins in the 0-day eggs than 0-day and 4- day eggs and

embryonated eggs. The protein bands which are stained yellowor brown may

considered as glycoproteins or lipoproteins. The glycoprotein and lipoproteins were in

increasing trend from 0- day eggs to eggs containing mature miracidia (Em) thus we

can say that basic protein changes in to different kinds of proteins during

development. The above discussion depends on the study of Dzandu et al, (1984);

they detected erethrocyte membrane proteins, sialoglycoproteins, and lipids in the

same polyacrylamide gel using a double staining technique.

A total of 10 and 9 polypeptides in the pH range of 3-10 were observed in the case of

G. explanatum and G. crumenifer. Some bands are common in both the species and

some bands are species specific. lEF demonstrates 17 bands from F. hepatica and 22

bands from F. gigantica between 3.5 to pH 10 and it was observed that some bands

were common in both the species, some are species specific (Allam et al, 2002). lEF

can be used in characterization of developmental stage-specific polypeptides but due

to lack of sample this could not be done in present study.

Transaminases: In the present study activity of transaminases (GOT and GPT)

was analysed in 0-day eggs, 4-day eggs, eggs containing mature miracidia (Em) and

adults of Gigantocotyle explanatum and Gastrothylax crumenifer. The results

obtained revealed that there is an increase in the levels of enzyme from 0-day eggs to

adult, in the 4-day eggs; eggs containing mature miracidia have highest enzyme

activities. The result suggests that these enzymes may play an important role in

protein metabolism, since 0-day eggs have lowest metabolic rate, so the activifies of

enzymes are lowest as the developmental process proceeds, metabolic rate increases

and the level of enzymes also increases. In a study of Min and Seo (1966) it was

56

found that larva! cestodes ha\e ver> lestricted activity of iransamination in

comparison to adult worms. They conclude that restricted transamination reaction in

larval tapeworm could be interpreted to be the result of limited growth and

reproductive phenomenon in the intermediate host.

In the present study the level of GOT was ft)und greater than GPT in adults of

G explanatum and G. crumenifer as well as their 0-day eggs, 4-day eggs and eggs

containing mature miracidia, it was supported by the studies of Min and Seo (1966),

that is their study activity of GOT was found consistently higher than GPT in the five

species of trematodes {Clonorchis sinensis, Paragonimus westermani, Faciola

hepatica, Eurytrema pancreaticum and Paramphistomum cervi) three species

cestodes (Dypylidium caninum, taenia pisiformis and Diphyllobothrium mansoni).

Further Siddiqui and Siddiqi (1990) also observed that activity of GOT was greater

than GPT in Gastrothylax crumenifer, Gigantocotyle explanatum and Echinostoma

malayalam. In the present study the activities of GOT were found higher in G.

crumenifer than G. explanatum whereas GPT activities are higher in G. explanatum

than G. crumenifer. It may be due to the difference in the habitate of the parasite.

Phosphatases: A phosphatase is an enzyme that removes a phosphate group from

its substrate by hydrolyzing phosphoric mono-esters into a phosphate ion and a

molecule with a free hydroxyl group. Two common phosphatases in many organisms

are alkaline and acid phosphatases.

During the present study the activities of alkaline and acid phosphatases

were recorded in 6-day eggs, 4-day eggs and eggs containing mature miracidia (Em)

of G. explanatum and G. crumenifer. On comparison the activity of phosphatases was

found to be highest in adult parasites in comparision to miracidial developmental

stages. In the developmental stages, eggs containing mature miracidia were found to

have highest activities. The increased enzyme activities may be correlated with the

progress in developmental stages, age and with morphological and physical changes.

Increase in enzyme activities during developmental process may also be an essential

requirement for organogenesis, embryonation and complex metabolic pathways to

overcome different environmental challenges during host finding. These enzymes

seem to play a role in embryo development. Previous histochemical work by Giboda

and Zdarska (1994) revealed alkaline phosphatase inside immature 5". mansoni eggs

associated with the vitelline membrane and the body surface of embryonating

57

miacidia. in mature eggs it was associated with miracidial germ ceils and sensory

endings of the neural cells. In the eggs of cestode Taenia taeniformis, alkaline

phosphatase activit> was found in the outer embr>'onic and onchospheral membranes,

where it seems to play a role in the development of embryo (Ajayvi et ah. 1985). In

adult S. mansoni, alkaline phosphatase is tightly bound to the tegumental membranes,

is immunogenic in infected patients, and has been used as a marker of infection (Pujol

et al, 1989). Increase in the activity from 0-day eggs to eggs containing mature

miracidia (Em) may be associated with biological roles of these enzymes like

processing of host proteins to fulfill nutritional requirements of the embryo, embryo

differentiation and in host parasite relationship.lt was supported by Cesari et al,

(2000) in their studies on "Enzyme activities in Schistosomsa mansoni soluble egg

antigen" because they concluded that these enzymes help eggs to pass through the

wall of the intestine or bladder and help miracidia to penetrate the smooth musle

extracellular matrix of snail vectors and in differentiation of embryo and it was also

suggested by Pino-Heiss et al, (1985) in their studies on '"Degradation of host

extracellular matrix by eggs and miracidia. In the present study highest enzyme

activity in adults may be due to many complex physiological activities like

gametogcnesis, fertilization and embryonation. The present study study supported by

the observation of Thorpe (1968) in his study on comparative enzyme histochemistry

of immature and mature stages of F. hepatica that apparent higher activities of

alkaline phosphatase were found in mature F. hepatica than immature form and no

apparent difference was found in acid phosphatase. Further acid phosphatase enzyme

activity was found to be greatest in third stage infective larvae and 8- day post

infection in adults of Nippostrongylus brasiliensis (Bolla et al, 1974).

Proteases: A protease (also termed peptidase or proteinase) is any enzyme that

conducts; proteolysis that is, begins protein catabolism by hydrolysis of the peptide

bonds that link amino acids together in the polypeptide chain forming the protein.

Proteases are classified into four major classes and are involved in a broad range of

eukaryotic processes. Proteases have also been found to play a number of critical roles

in the virulence of pathogenic agents, particularly of nematode parasites. Parasitic

proteases are involved in different aspects of host-parasite interactions. They facilitate

the invasion of host tissues and allow nutrition as well as the survival of the parasite

in its host. Proteases also participate in the parasite's evasion from the host's immune

58

response. The functional diversity and complexity of these enzymes were described in

the review by Irap and Boireau (2000) with a particular focus on the proteases of four

helminths: Schistosoma sp.. Fasciola sp., Taenia sp. and Haemonchus sp. Some of

these proteases, especially the cysteine proteases secreted by the parasitic trematode

Fasciola hepalica, have been successfully tested in experimental immunodiagnosis.

Proteases identified in helminth parasites are proposed as major potential targets for

immunotherapy and chemotherapy against parasitic diseases (Trap and Boireau,

2000).

In the present study various classes of proteases in 0-day eggs, 4-day eggs and

eggs containing mature miracidia (Em) of G. explanatum and G. crumenifer were

analyzed by SDS-PAGE using gelatin copolymerized gel electrophoresis. The gels

stained for protease activity showed complex array of proteases with varying

molecular weights. The results showed that number of specific bands in eggs

containings mature miracidia was highest in both G. explanatum and G. crumenifer,

showing increased proteolytic activity during the development. In the present study 0-

day eggs of G. explanatum did not show any protease activity while in 4- day eggs 5

protease bands with a molecular weight range of 53-123 kDa and one specific band of

39.5 kDa were resolved. Further in eggs containing mature miracidia 6 bands of

proteases were resolved in a molecular weight range of 53-123 kDa having two

specific protease bands with an apparent molecular weight of 120 and 60 kDa but in

the adult only two protease bands were identified of molecular weight 123 and 35.9

kDa with one specific protease band of molecular weight 35.9 kDa. In G. crumenifer,

0-day eggs revealed one protease band with an apparent molecular weight of 123 kDa,

whereas in 4-day eggs 5 bands were resolved in a molecular weight range of 30-123

kDa with one specific band of 120 kDa and in the eggs containing mature miracidia

stage 7 protease bands were observed in a molecular weight range of 30-123 kDa with

3 specific protease bands of 108, 37, 34 kDa. In adult of G. crumenifer only two

bands were resolved of molecular weight 123 kDa and 78.6 kDa with one specific

protease band of molecular weight 78.6 kDa. The heterogeneity and increased number

of protease bands in the eggs containing mature miracidia (Em) as compared to zero

day and 4- day eggs may be associated in prepararing the miracidia to penetrate snail

tissue and in the establishment of host parasite relationship. Microfilaria! lysate of

Bnigia malayi showed three protease molecules of 40 kDa, 180 kDa, and 200 kDa,

59

the L3 larval lysate had 6 protease molecules of 13, 25, 37, 49, 70, and 200 KDa size

(Bhandarv et al. 2006). They concluded that heterogeneity in the composition of

proteases in mf lystate and L3 larval stages might be appropriate, considering the

different host environments to which they are exposed. The increase in the number of

protease bands during development from 0 day eggs to eggs containing mature

miracidia (Em) may have great physiological roles. Several investigations studied the

proteases during development of parasites such as: the sexine proteases from larvae of

Heliothis virescence (John-Ston et al., 1995), the neutral proteases of three

developmental stages of S. mansoni (Auriault et al ,\ 982). serine proteases from

Chrysomya hazziana larvae (Musharsini et al, 2000), a neutral thiol protease from

Paragonimus westermani metacervariae (Yamakami and Hamajima, 1990) and a

serine and cystein type proteases from Eimeria tenella oocytes (Michalski et al,

1994). Enzymatic assay have shown that F. hepatica cathepsin L activity is expressed

in different developmental stages (Carmona et al, 1993; Smith et al, 1993).

Generally, different forms of proteases were demonstrated in different stages of

parasites (Siddiqui et al, 1993; Ghoneim and Klinkert, 1995; Johnston et al, 1995;

Hawthrone et al, 2000; Muharsini et al, 2000; Harmsen et al, 2004). Protease

activity was detected in the culture medium of transforming miracidia and in

detergent extracts of Schistosoma mansoni miracidia and primary sporocysts

(Yoshino, et al, 1993). Since no published report on the developmental stage -

specific proteases of aniphistome parasites is available therefore the present study

related to proteases in developmental stages is important and a similarity can be

correlated with Schistosoma spp. and Fasciola hepatica. The activities of proteases

during the miracidial development in F. gigantica increased gradually (from 0-12 day

eggs) and a significant increase in protease activity was detected on day 15 (mature

miracidium) (Mohamed et al, 2005). Proteolytic enzymes secreted from several life

cycle stages of S. mansoni have been implicated in a variety of fiinctions including

adult worm nutrient acquisition (Zerda et al, 1988), cercarial skin penetration and

movement of eggs through host tissue, (Mc Kerrow and Doenhoff, 1988).

Schistosome miracidia also posses proteinase activity (Pino-Heiss et al, 1985; Sung

and Dresden, 1986) that appears to be located in the lateral penetration glands

(Dresden et al, 1983). Pino-Heiss et al, (1985) discussed that both eggs and

miracidia secrete proteinase which may capable of degrading at least the glycoprotein

60

components of extracellular matrix to facilitate their migration through intestinal wall

or penetration of host tissue/Thus in the case of present study it can be concluded that

upon release, proteinase ma) play a role in snail tissue penetration, larval nutrient

acquisition or in the modulation of internal defense responses to invading larvae.

Glutathione S-transferase: Glutathione S-transferases are a large family of

multifunctional dimeric enzymes that conjugate reduced glutathione to electrophilic

centers in hydrophobic organic compounds. The GST enzymatic activity has been

described in the adult and larval stages of helminthes Joachim and Ruttkowski (2008).

Several forms and isoforms of the enzyme have been purified and GST genes have

also been isolated and expressed as recombinant proteins (Vibanco- Perez and Landa-

Pierda 1998). The helminth GSTs participate in detoxification of lipid hydroperoxides

and carbonyl cytotoxics produced by oxygen-reactive intermediates (ORI). The ORIs

can come from the endogenous parasite metabolism or from the host immune system.

The helminth GSTs are able to conjugate glutathione to xenobiotic compounds or to

bind to anthelmintics drugs. GST is usually localized near to host-parasite interface.

This enzyme has been identified as potentially vulnerable targets in immunotherapy

and chemotherapy (Vibanco- Perez, and Landa- Pierda, 1998).

In the present v»'ork GST activity was determined spectrophotometrically as

well as various isozymes of enzyme were separated by native gel to identify stage-

specific differences in 0-day eggs, 4-day eggs, eggs containing mature miracidia and

adult of G. explanatum and G. crumenifer. The results revealed an increase in the

activity of enzyme and also number of isozymes in gel as the development proceeds.

There were 3, 3, 5 and 5 bands of GST isozymes in the 0-day eggs, 4-day eggs, eggs

containing mature miracidia (Em) and adult stages respectively having 0, 0, 2 and 4

specific GST bands with an apparent molecular weight of 19 kDa and 17.5 kDa (for

eggs containing mature miracidia), 118 kDa, 104 kDa, 52 kDa, 41 kDa (for adult). In

G. explanatum 3, 4, 6 and 6 isozyme bands were found in 0-day eggs, 4-day eggs,

eggs containing mature miracidia (Em) and adult stages respectively with 0, 1, 2 and 2

specific GST bands and apparent molecular weight 78 kDa (for 4-day eggs) 22.5, 19

kDa (for eggs containing mature miracidia) and 91, 25 KDa (for adult). GST is a

multifunctional protein so different isozymes are resolved. Variation in GST activity

and their isozyme pattern occurred during different stages of the parasite understudy

and an increase in enzyme activity was found from 0-day eggs to adult stage. Greatest

61

eriZ}'me activiU in aduit and in the miiacidial deveiopmeutai stages highest enz>me

activity m eggs containing mature miracidia may be due to survival of the miracidia in

the snail tissue and adult parasite in the mammalian host environment because GST is

an antioxidant enzyme and work as the major detoxifier enzyme. It may also due to

high biosynthetic activit> during the development of miracidia and adult, during the

development and growth, a large capacity for detoxification is needed to prevent

xenobiotic damage to many sensitive, biosynthetic processes that are occurring and to

remove endogenous toxic compounds that might accumulate. Joachim and

Ruttkowski (2008) found that there was a difference in the enzyme activity in

different stages of Oesophagostomum dentatum and highest activity was found in

third stage larvae, they concluded that chages in levels of enzymes could be host-

induced stress, changes in growth rate and metabolism or changes in the utilization of

energy sources with subsequent production of endotoxic metabolites, they also found

that GST was higher in females than in males they concluded that this difference may

be related either to specific function of GST in this stage or to expression of this

enzyme in the maturating egg since the majority of the females were fertilized at

harvesting. In a similar study, the activities of GST increased throughout larval

development of mosquito, Aedes aegypti and reached a maxium in the metamorphosis

stage (Hazelton and Calvin, 1982). GST was detected in the eggs; it increased

throughout the larval stage and was the highest in two day-old fifth instar larva

(Rajurkar et al., 2003). GST has been detected in a range of helminthes (Brophy and

Barrett, 1990) and are implicated in a variety of metabolic pathways by conjugation of

glutathione to different agents, including detoxification of anthelmintic substances

and intracellular transport (Precious and Barret, 1989; Sharp et al, 1991; O' Leary

and Tracy, 1992; Miller et al., 1994; Kampotter et al. 2003; Leirs et al., 2003). The

GSTs have attracted attention due to their high level constitutive expression and

antigenicity and their potential role as vaccine targets (Mitchell, 1989; Sexton et al.,

1990; Brophy and Pritchard, 1994; Barrett, 1995; Campbell et al., 2001). The

majority of helminthe GSTs characterized to date is of cytosolic origin and comprises

the sigma (or alpha) class. Trematodes (first of all. Schistosoma mansoni and

Schistosoma japonicum as well as liver fluke Fasciola hepatica) were in the focus of

GST research (Gupta and Srivastava, 2006). There is no report on developmental

62

stage -specific GSTs of amphistome parasites so this study with further investigation

ma}' lead to the development of vaccine and drug against aniphistomiasis

f rom the present study it can be concluded that as amphistome parasites are

passed through several developmental stages and most stages are parasitic and this

parasitic mode of life necessitates the development of an efficient metabolism as a

biochemical adaptation because of rapid turn over and enormous fecundity to ensure

establishment of germ line. The adaptations involve biosynthetic processes, energy

production, and detoxification of metabolic wastes through biodegradation process

and establishment of host parasite interaction. During the chain of these events a

bewildering array of enzyme activity participates to catalyze these reactions. In fact

enzymes can be regarded as the key of metabolism that unravels the mysteries of

various metabolic idiosyncracies. The development is controlled by sets of genes

which are activated or suppressed on receiving an appropriate stimulus involving

complex interaction between extrinsic and intrinsic factors at a specific stage of the

life cycle. Such controlled changes may also be responsible for the differential

antigenic and protein turnover in parasites. The present study will help in identifying

stage-specific enzymes and proteins and in future potential of such stage specific

en^ym.es and proteins can be used as targets for immuno- and chemotherapy.

In future studies, partial purification and characterization of polypeptides

obtained from adult, miracidial and intra-molluscan larval stages of Gastrothylax

crumenifer and Gigantocotyle explanatum will be carried out. A 2D map will be

generated using proteomic approach with the aim to find and exploit some stage-

specific unique target to control the larval, immature and adult stages.

63

.EMCE

m

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