Characterisation of Antigens of

Hookworm Parasites

( SUMMARY )

A DISSERTATION SUBMITTED

TO THE

ALIGARH MUSLIM UNIVERSITY, ALIGARH

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

IN

BIOCHEMISTRY

BY

SHAIINI MOHAN M. Sc. (Biochem. ) ,M. Phil.

BIOCHEMISTRY DIVISION

CENTRAL DRUG RESEARCH INSTITUTE LUGKNOW-226 00I

AUGUST-1990

Ancylostomiasis is one of the major public health problems

in tropical countries. The world incidence of hookworm infect­

ions are 1000 million with the death rate of 55 thousand per

year. The currently available anthelmintics are not very

effective. This clearly emphasises the need to intensify

the control measures in hookworm endemic areas . An alternate

approach for the permanent control of the disease would be

to develop immunoprophylactic measures against hookworm

paras i tes . However, the existing knowledge about the molecular

immunology of hookworm infection is st i l l in i ts infancy.

Hookworm parasites evoke functional protective immunity and

some reports are available regarding the protective potential

of live parasit ic stages as well as of somatic extracts and

excretory-secretory products from adult and larval stages,

however, not much is known about the molecules/antigens which

are involved in evoking these protective responses. A basic

knowledge about the total antigenic composition of the para­

sites is essential not only for understanding the host parasite

relationships but also for the identification of antigenic t a r ­

gets of protective immunity. • We have, therefore, directed

our studies towards the characterisation of antigens from adult

and larval stages of human hookworm parasi te ; Ancylostoma

ceylanicum, which has earl ier been used for the screenings

of anthelmintic compounds by several workers. Total protein

and antigenic composition of adult and larval stages of

A. ceylanicum were studied employing SDS-polyacrylamide gel

electrophoresis (SDS-PAGE) and immiinoelectrophoretic techniques

using hyperimmune rabbit sera. The levels of parasite specific

antibodies in immune sera were determined by enzyme linked

immunosorbent assay (ELISA). Similar techniques were used

for antigenic analysis of the excretory-secretory products

from adult A. ceylanicum. The protective potential of somatic

extracts from adult and larvae as wellas the excretory-secretory

products from adult A.ceylanicum was also studied by immunising

the naive hamsters with these antigens (somatic extracts from

adult and larvae and excretory-secretory products from adults)

followed by challenge infection.

The somatic extracts from adult (AcA) and larval stages

(AcL) of A. ceylanicum were separated on SDS-polyacrylamide

gradient gel electrophoresis (SDS-PAGE), which showed the

presence of 47 protein bands m adults and 43 in Larvae m

the molecular weight range of 10-170 kD. Most ot thr f)rr)tfin

bands were found to be common between these two stages

(adult and la rvae) , however, 8 (130, 125, 42, iO, 20, 17,

16.5 and 10 kD) and 4 (170, 160, 153 and 85 kD) protein

bands specific to adult and larval stages respectively were

observed. The quantitation of parasite specific antibodies

in immune rabbi t sera was done by enzyme linked immunosorbent

assay and reciprocal antibody t i te rs of 256,000 and 128, 000

were obtained for rabbit anti-adult and rabbit anti-larval

sera respectively using AcA antigen at optimum concentration

of 0.25 ug/well. Both these sera showed the reciprocal anti-

body t i t e r s of 256,000 with AcL at optimum concentration

of 0.50 ug/well.

The antigenic analysis of somatic extracts from adult

and larval stages of A.ceylanicum was init ially done by Immuno­

electrophoresis ( lEP), which revealed 10-12 precipitin lines

in AcA with rabbit anti-AcA serum and 6-7 precipitin bands

in AcL with rabbit anti-AcL serum. Further characterisation

of these somatic antigens was carried out employing crossed

Immunoelectrophoresis (CIE) which showed the presence of

34 antigenic components in AcA and 19 antigens in AcL. Out

of 34 AcA antigens, 25 were anodic and 9 cathodic while 17

anodic and 2 cathodic antigens were observed in AcL. In order

to study the cross-reactive or common as well as stage/parasite

specific antigens of A.ceylanicum, crossed Immunoelectrophoresis

employing heterologous sera and crossed line immunoelectro-

phoresis (CLIE) were used. When AcA and AcL were reacted

with heterologous immune rabbit sera in CIE, most of the

antigens were found common or cross-reactive between the

two stages, however, 12 antigens specific to adult and 5 anti­

gens specific to larval stage were observed. The subsequent

analysis of CLIE also revealed the same number of common

or cross-reactive as well as stage specific antigens in AcA

and AcL. In order to analyse A. ceylanicum antigens having

cross-react ivi ty with rat hookworm - Nippostrongylus brasi l iensis ,

CIE using immune rabbi t sera raised against adult (NbA) and

infective larval (NbL) somatic extracts of N, brasil iensis and

CLIE using NbA and NbL in the in te rmedia te ge l , were done.

Most of the antigens (22-26) were found to be AcA spec i f i c ,

only 12 and 8 antigens of AcA were found to be common/cross

r eac t i ve with NbA and NbL r e s p e c t i v e l y . On analysing the

c r o s s - r e a c t i v e or common antigens of AcL with NbL and NbA,

13 common or c ross r e a c t i v e and 6 AcL speci f ic antigens were

o b s e r v e d .

Another set of antigens analysed in t h e p resen t s tudy

i s the e x c r e t o r y - s e c r e t o r y (E-S) p roduc t s from adult

A. ceylanicum . The condit ions for the p repara t ion of E-S

p roduc t s were opt imised with r e s pec t to medium as well as

the incubation t i m e . Out of t h r e e different media; RPMI-1640,

DMEM and R inge r ' s solution t r i e d , R inge r ' s solution proved

to be the bes t as the mot i l i ty of the worms was maximum

in t h i s medium and the worms could be maintained upto 6h

without changing the medium. The amount of pro te in re leased

was found to be 95±15 ug/100 adults in RPMI-1640 and DMEM

as compared to 125±25 ug/100 adult worms in R inge r ' s solut ion.

Immunoelectrophoret ic ana lys i s using r a b b i t anti-AcA serum

showed the presence of 5-7 p r e c i p i t i n bands in E-S products

p r e p a r e d in t h e s e media . As t h e r e was not any significant

difference in the amount of pro te in r e l ea sed as well as the

antigenic p a t t e r n s of E-S p roduc t s p r e p a r e d in t h r e e media ,

R inge r ' s solution (containing s a l t s and glucose) was chosen

to obtain the E-S p roduc t s from A.ceylanicum adu l t s because

t h i s i s much s imple r and more economical in comparison to

o the r two media (RPMI-1640 and DMEM).

A polyvalent hyperimmune rabbit serum was raised against

A. ceylanicum adult E-S products and the t i ter of parasite

specific antibodies in immune rabbit sera was determined

by enzyme linked immunosorbent assay using optimum concentrat­

ion of 0.50 ug E-S antigen/well. A reciprocal antibody t i ter

of 256,000 N*as obtained with rabbit anti-E-S serum. The

E-S antigens were also tested with the immune rabbit sera

raised against somatic extracts of A.ceylanicum in ELISA and

a t i ter value of 128,000 was obtained for both rabbit anti-

adult and rabbit anti- larval sera . Crossed Immunoelectropho­

res is (CIE) using rabbit anti-E-S serum revealed 16 antigens

in A. ceylanicum E-S products, out of which 13 were anodic

and 3 moved towards the cathode. E-S products have also

been found to share antigenic determinants with the somatic

extracts from adult and larval stages. lEP analysis of E-S

products showed the presence of 6 and 4 precipitin bands

with rabbit anti-AcA and rabbit anti-AcL sera respectively.

On further analysing the E-S products by CIE using rabbit

sera raised against somatic extracts 12 and 7 antigens of

E-S products were found to share antigenic determinants with

AcA and AcL respect ively . Tandem crossed immunoelectropho-

resis (TCIE) of E-S products with AcA and AcL also revealed

almost the same number of common/cross-reactive antigens

between the E-S products and the somatic ex t rac ts . The pre­

sence of host serum proteins in the E-S products was also

analysed by CIE using rabbit serum raised against normal

6

hamster serum, and 6 antigens in the E-S products appeared

to be of host origin.

The protective potential of somatic extracts from adult

and larval stages as well as excretory-secretory products

from adult A.ceylanicum was tested by immunizing the hamsters

with these antigens and evaluating the immunized hamsters

for protective immunity. The sera obtained from immunized

hamsters, before challenge, showed significant levels of para­

site specific antibodies. The immunodiffusion and immuno-

electrophoretic analysis revealed the presence of precipitin

antibodies in immune hamster sera and 7 precipitin bands

in adult somatic extracts and 4 in both larval somatic extracts

and E-S products. The hamsters immunized with larval somatic

extracts showed maximum protection (93% reduction in worm

burden) while 75% and 63% protection was observed in hamsters

immunized with excretory-secretory products and adult somatic

extracts respect ively.

Therefore, in the present study we have been able to

characterise the protein and antigenic components of adult

and larval stages of A.ceylanicum using hyperimmune rabbit

sera having high t i te rs of parasite specific antibodies and

certain biochemical and immunochemical techniques. The common

or cross-reactive antigens between the two life stages of

A. ceylanicuiD and also between the two hookworm parasites

(A.ceylanicum and N.braslliensls) as well as stage and parasite

specilic antigens have also been identified. We have been

able to prepare the excretory-secretory products of A.ceylanicum

adults using Ringer's solution which is much simpler and

a more economical medium. Antigenic characterisation of E-S

products revealed less complex nature of these products as

compared to the a-dult SQma.tic extracts» The protective potea-

t ials of the somatic extracts from adult and larval stages

and also of E-S products from adult A. cey lanicum was also

studied. The larval somatic ext rac ts , though contain less

number of antigens, showed maximum protection followed by

excretory-secretory products and adult somatic extracts .

These studies provide basic information about the antigenic

compositions of gomatic extracts and excretory-secretory pro­

ducts of A. ceylatnicum as well as the comparative efficacy

of these antigens in inducing protective immunity against this

paras i te . However, further studies on molecular characterisa­

tion of these antigens are certainly required for identifying

the antigenic targets of protective immunity.

Characterisation of Antigens of

Hookworm Parasites

A DISSERTATION SUBMITTED

TO THE

ALIGARH MUSLIM UNIVERSITY, ALIGARH

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

IN

BIOCHEMISTRY

BY

5Hi4LINI MOHAN M. Sc. (Biochem. ) ,M. Phil.

BIOCHEMISTRY DIVISION

CENTRAL DRUG RESEARCH INSTITUTE

LUGKNOW-226 001 AUGUST-1990

T4022

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ACKNOblLEVGEUENTS

I takd thd piiviiZQH to KZcoid my thanks to Vn.. A.M.Siddiqui,

Pn.o{ie.Mofi, dtpaitmant o^ Biochzmi^tfty, Migaih Uu^tim Univzi^ity,

AZigafih (^o^i hi^ gaidanct and hzlp.

I exp^e4-6 thz deep -ien^e oi$ gratitude, to P- .lM- ) W.A.

Kaa-iihal, Stiznti^t, ViviMon o($ Biothzmiit^y and V^. V.C. KaU'6ha-£,

Sciznti^t, Viviiion 0($ Hicfiobiology, CVRl, Lucfenow, ^oi thzii

az^thztic. 4ugge4-t-con4, in^piiing gixidanaz and {,Kult{^al tfiiticium

through u)hich thi^ paramount ta-ik could acqulKZ thz p^e^ew-t

ihapz.

Uy gtatz^ainz^^ -6 ai'do duz touoaA-d^ Vn.. B.N. Vhawan,Vi'iZctoi,

CVRl, Vi. Pizm Sagai, Hzad, VzpafitmZnt 0($ Biochzmi4>tiy and thz

collzaguZ'ii 0($ thz dzpan-trnznti o($ Biochzmi'i^tfLy and Vaia-ftitotogy

who havz hzipzd me duiing thz zompZztion Oj$ thi'i> di^iy^zitation.

I am indzbtzd to Council o{^ Scizntif^ic and Inda-iit'ilal

Rz^za^ch, Wew Vzlhi (Jo-t financial Mppo^t. Thz Irmaculatz typing

oi Mfi. K.L. Gupta -c4 al^o acknooolzdgzd.

Lastly, but not thz Iza^t, I havz no itioid^ to zx.piZM my

humblz giatitudz, pKoiound fizgaid^ and lovz to my paiznt^ ^on.

thzin i^oibzaiancz and a^^zctionatz blzMing^ which uiziz all

thz uoay.

ShcUinJUofutifr

C O N T E N T S

Preface

Page No.

i - i i

Abbrevia t ions . . . . . . . . . i i i

Chapter I

Introduction and Review of L i t e ra tu re . . . . . . 1 - 2 1

Chapter II

Mater ia ls and Methods . . . . . . 2 2 - 3 2

Chapter III

Results and Discussion . . . . . .

Section I

Antigenic ana lys i s of somatic e x t r a c t s from adul t and infect ive l a r v a l s tages of Ancylostoma ceylanicum . . . 3 3 - 7 5

Section II

Excre to ry -Sec re to ry p roduc t s from adult

A.ceylanicum 76 - 106

Chapter IV

Summary and Conclusion . . . . . . 107 - 114

Bibliography . . . . . . . . . 1 1 5 - 1 2 5

* * * * * * * *

*

( i )

P r e f a c e

Almost every individual in the tropical regions of

the world is suffering from parasit ic infections of one kind

or the other . According to some recent repor ts , millions

of people are affected by differnt helminth parasites including

hookworms which account for 1000 million cases annually.

Heavy burdens of hookworm parasi tes cause severe anaemia

which may sometime prove fatal and can also lead to in­

tellectual and growth retardation. Recently, chemotherapy

targetted to heavily infected individuals has come out as

a real is t ic approach, however, the permanent control measures

of the disease are s t i l l lacking. Hence, there is an urgent

need to evolve an alternate strategy for controlling the hook­

worm disease. The existing knowledge about the molecular

immunology of hookworm infection is very poor and characteri­

sation of hookworm antigens may provide valuable leads about

the antigenic moelcules of the possible immunoprophylactic

and immunodiagnostic significance.

The studies reported here are directed towards the

characterisation of somatic as well as excretory-secretory

antigens of Ancylostoma ceylanicum - a human hookworm, which

has been used for chemotherapeutic screening of potential

anthelmintics in our Institute. The antigens from adult and

infective larval stages of A. ceylanicum were also analysed

in order to identify the common/cross-reactive and stage/

parasite specific antigens of A. ceylanicum. The potential

of these antigens (somatic extracts from adult and larval

( i i)

stages as well as of excretory-secretory products from adult

A.ceylanicum) in inducing protective immunity in experimental

animals to challenge infection was investigated and the larval

somatic extracts showed significant degree of protection. The

information obtained from these findings may be of great impor­

tance for the identification of antigens of possible protective

potential.

A B B R E V I A T I O N S

( i i i )

AcA

AcL

CIE

CLIE

ELISA

E-S

Ig

lEP

ID

"2°2

ug

NbA

Nbl,

OPD

SDS-PAGE

TEMED

TIE

A.ceylanicum adul t somatic e x t r a c t

A.ceylanicum l a r v a l somatic e x t r a c t

Crossed-immuno e l e c t r o p h o r e s i s

Crossed l ine Immunoelectrophoresis

Enzyme l inked immunosorbent assay

E x c r e t o r y - s e c r e t o r y

Immunoglobulin

Immunoelectrophoresis

Immunodiffusion

Hydrogen p e r o x i d e

Infective l a r v a l stage

microgram

N . b r a s i l i e n s i s adul t somatic e x t r a c t

N . b r a s i l i e n s i s l a r v a l somatic ex t r ac t

0 -phenylene diamine

SDS-polyacrylamide gel e l e c t r o p h o r e s i s

Tetra e t h y l methylene e thy lene diamine

Tandem-crossed Immunoelectrophoresis

CHAPTER I

INTRODUCTION AND

REVIEW OF LITERATURE

1

Hookworm parasites are among the imoortant intestinal

nematodes whose prevalence and concomitant detrimental effects

on the body functions have made them to be ranked as one

of the important helminthic diseases of man in developing

and underdeveloped countries. Although the infection does

not cause mortality, it saps the vital i ty of the hosts already-

suffering from malnutrition. According to the recent repor ts ,

about 1000 million people may be infected with hookworm

parasites (Hotez ^ al^.. 1987; Crompton, 1989).

"Hookworm disease", in man is mainly caused by the

three species - Necator americanus, Ancylostoma duodenale

and Ancylostoma ceylanicum. Besides the above three human

pathogens, the other parasites pathogenic to livestocks are

A.caninum (dog), A.tubaeformae (ca ts ) , Nippostrongylus

brasil iensis ( r a t s ) , Nematospiroides dubius (mouse), Bunonstomum

trigonocephalum (sheep and goat), B.phelabotomum (cow) and

Gaigeria pachycetis ( sheep) .

Hookworm infection is more prevalent in tropical and

subtropical regions of Asia, Africa, Europe and America. In

Asian continent the most endemic countries are India, Malaysia,

China and Japan. In India, the tropical climate, poor sanitation

and poorer health education favour the growth, multiplication

and dissemination of all parasit ic infections including hook­

worms. The morbidity and mortalitv caused by the disease

were first reported in tea plantations of Assam, where approxi­

mately 90% of the population harbour this infection. The inci-

dence of the disease is also very high in plain where p^ddy

cultivation is predominant (Raghavan, 1967), while the incidence

is low in Uttar Pradesh and Pondicherry (Yunus, 1977; Singh

e^ al^., 1978).

The period from infection to the first appearance of

eggs in the faeces of the host is constant for each species

of hookworm. The ovum undergoe," four moults and second

moult produces an infective stage i . e . the thi rd stage larvae,

L . . This stage after penetrating the skin moves towards lungs

and the th i rd moult takes place. L. larvae with a provisional

buccal capsule, enters the larynx, then moves to pharynx,

oesophageous and finally settles down in the small intestine.

The migratory phase occupies about 7-15% of the prepatent

period. The last moulting occurs in the intestine, the adults

mature, mate and reproduce and thus the cycle continues. Among

human hookworm paras i tes , the shortest prepatent period has

been observed in case of A.ceylanicum (18-26 days) , while

the prepatent period for A.duodenale and N.americanus is longer.

Maximum egg output was observed between 12 and 18 months

of A.duodenale infection in man and the life span of the worms

was 6-7 years (Kendrick, 1934), while that of N.americanus

was reported to be 5-6 years .

There are no specific signs and symptoms in the vast

majority of cases of hookworm infection and their order of

appearance are closely related to the chronology of migration

and development of the paras i tes . The symptoms can con-

Third stage Infective larvae

(L3)

Blood circulation (Ground Itcti)

Eggs In faeces

SOIL

>

Adult Intestine (Anaemia,abdominal, discomfort)

Stomach

Tracheolarynx (Bronchitis, Asthma)

/

Fig . 1: General l i fe cyc le of hookworm paras i te .

veniently be divided into two; those associated with migration

of L and presence of adult worms are primary ones, while

the secondary symptoms are the consequences of physiological,

biochemical and haematological disturbances that follow the

persistence of intestinal infection and constitute what is commonly

known as 'hookworm d i sease ' . A primary heavy infection

will induce ground itch followed by fever, asthmatic wheezing

and a high degree of eosinophilia which will be self limiting.

In later stages, the ancylostomiasis is connected with iron

deficiency anaemia associated with epigastric pain or discomfort

which may be relived by food and mistaken for duodenal ulcer.

The functional consequences of th is disturbed iron-balance

and impaired nutritional status include growth retardation,

reduced working productivity and a potential adverse effect

on learning and cognitive development. Severe intestinal haemo­

rrhage due to the infection might lead to a drop in haemoglobin

(Hb) level as low as 2 g/100 g of blood and in erythrocyte

count to below 1000/ml. Chronic and acute, both kinds of

anaemic conditions are observed in the patients of hookworm

infection. Numerous secondary signs and symptoms attributable

to anaemia and hypoproteinaemia accompany chronic hookworm

infection. The most important of these are associated with

progressive cardiac insufficiency and congestive failure. A

wide range of reproductive disorders have also been attributed

to the effects of hookworm infection.

Chemotheraoy based on efficacious anthelmintic drugs

like-Mebendazole, Thiabendazole, Bephinium, Levamisole ,

Tetrachloroethylene etc . remains the starting point of a modern

control programme against hookworms (Crompton, 1989). However,

these drugs are effective against the adult stage of the para­

site and repeated doses cause various types of side effects.

Hence, there is an urgent need to evolve an alternate strategy

which should be effective against the infective larval stage

and can check the infection as soon as it occurs. A realistic

approach would be to diagnose the disease at an early stage

as well as to develop immunoproohylactic measures, which

require a good understanding of the molecular immunology of

hookworm infections. These studies will certainly provide

the basis for rational vaccine development as well as for immuno-

diagnosis of the disease. Accordingly, a survey of literature

was made along these l ines.

REVIEW OF LITERATURE

The efforts to understand the molecular and immuno­

logical processes underlying hookworm infection have greatly

been limited due to the lack of suitable animal models. Most

of the JjT vivo studies for examining the complete life cycle

of Ancylostnma hookworms require the use of dogs or cats,

of course with the exceotion of some studies which were done

in primates. Most of the studies have been done using these

animal models but the expense and the time required to maintain

the parasites m these laboratory hosts has hampered the studies

on hookworm paras i tes . An important development has been

the establishment of human hookworms in laboratory rodents

so that controlled experimental studies can be made on these

paras i tes . The maintenance of A.ceylanicum m golden hamsters

(Carroll and Grove, 1984) and of N.americanus m neonatal

hamsters (Behnke et_ al^., 1986) has opened up new vistas for

laboratory investigations. This achievement has facilitated

work on antigen characterisation, immunity and vaccine develop­

ment. The role of surface antigens and excretory-secretorv

products which include certain metabolic enzymes like proteases,

IS st i l l not well establ ished. By using modern techniques,

these antigens can be well studied and it will help m develop­

ing better immunoprophylactic and immunodiagnostic measures.

Studies on immunology of hookworm parasites have mainly been

directed towards the two major aspects - one is immunodiagnosis

dealing mainly with serological or host immune responses and

the other aspect is protective immunity.

Immunodiagnosis

The development of immunodiagnostic methods for hook­

worm infection has received a l i t t le attention m view of the

relative ease of diagnosis by faecal examination, but, in fact,

the hookworm eggs may be sometimes misunderstood with the

other intestinal helminth paras i te . Hookworm parasites have

been shown to mediate a wide v a r i e t y of serological react ions

in v i t r o , de tec ted by complement fixation (Magaudda Borzi

and Penn is i , 1958), gel diffusion (Singh, 1965), la tex flocculation

(Rombert et_ al^., 1967); haemagglutination (Desowitz, 1967:

Jayawardene and Viiayaratnam, 1968; Ball and Ba r t l e t t , 1969),

immunoprecipitat ion (Yamanaka, 1960; Ball and Ba r t l e t t , 1969;

Ogilvie et^ al^., 1978) and immunofluorescence (de Azevedo et

a l . , 1971; Lewis et_ al^., 1978). A s tudy of immunological r e ­

actions during exper imenta l infection of N.americanus was done

long back by Ball and Bar t le t t (1969) . Later on, Poulain et_

a l . (1976) s tudied the local sys temic responses in r a t s infected

with N . b r a s i l i e n s i s by determining t h e an t ibody t i t e r s of both

se ra and in tes t ina l mucosa using p a s s i v e haemagglutination t e s t

and found tha t a cons iderab le port ion of a c t i v i t y was associa ted

with IgA f rac t ion . Specific IgA and o the r i so types of ant ibody

to N . b r a s i l i e n s i s antigens have a lso been de tec ted in sera as

well as in tes t ina l secre t ions by seve ra l o the r workers (See

see et^al_., 1976; Sinski and Holmes, 1977; Brown et_ al_., 1981a,b) .

People have t r i e d to quant i ta te the an t ibody t i t e r s during the

course of infection employing var ious immunological techniques

l ike counter current immunoelect roohores is (Bhopale et_ a l . ,

1981; Men on and Bhopale , 1985), enzyme l inked immunosorbent

assay (Ogilvie et^ al^., 1978) and radioimmunoassay (Sinski and

Holmes, 1978). Counter current Immunoelectrophoresis has been

shown to have a ve ry l imited appl ica t ion in serodiagnos is because

of mult iple helminth infections as well as c ros s - r eac t ions among

var ious hookworm s p e c i e s . Radioimmunoassay has successfuUv

been used for quantifying the specific antibody responses in

rats infected with N.brasiliensis and it was found to be of

local IgA type (Sinski and Holmes, 1978). It has also been

reported by the same workers that the local specific IgA anti­

bodies were closely associated with immunity against gastro­

intestinal nematode. The local antibody responses (IgA, IgM

and IgG levels) in rats parasit ised with N.brasiliensis infection

were investigated by measuring the levels of coproantibodies

(Wedrychowicz et_ al_., 1983), Recently, the usefulness of radio­

immunoassay and reverse enzyme immunoassay using excretory-

secretory products from hookworm parasites has been demons­

trated in serodiagnosis and prognosis of hookworm infections

(Ganguly et_ al^., 1988).

The hookworm infections have been shown to evoke

immediate type hypersensit ivi ty reactions as demonstrated by

intradermal tests conducted with antigenic preparations from

various hookworm parasites (de Hurtado and Layrisse, 1968).

The sensitivity of skin reaction was found to be 80% or even

more and some results indicated that the intensity of skin

reaction may be related to the intensity of hookworm infection

(Lobel et_ al^., 1968). In support of these findings a positive

relationship has been shown between the skin reaction (diameter

of the wheel formed) and the level of infection (faecal egg

count) by some workers (de Hurtado and Layrisse, 1968; Lobel

et al_. ,1968). A significant correlation between the wheel size

and eosinophillia and presence of signs and symptoms of hook-

worm infection has also been shown by Ishizaki et a l . (1961).

Antigens used for the skin test include protein or polysaccharide

fractions prepared respectively by t rypt ic digestions of larvae

or adult hookworms (Noda, 1953; Sawada et_ al^., 1954; Wei

and Kuo, 1958; Shih et al^., 1959; Prasad and Mathur, 1962)

or by aqueous extraction (Yamanaka, 1960; Ishizaki et_ a l . ,

1961; de Hurtado and Layrisse, 1968; Lobel et a]_., 1968). Skin

test has been found to be positive in man using antigens from

canine hookworm suggesting the cross reactivity among these

antigens (Sawada et_ al^., 1954, 1961; Yamanaka, 1960). It has

been shown that the sera obtained from hookworm or ascariasis

patients also cross-react well with cestode antigens (Correa-

Oliveira et al^., 1988). The demonstration of these cross r e ­

actions points out the problems of serodiagnosis. The heat

shock proteins have been shown to have better prospects for

a species specific diagnostic test (Newport et_ al^., 1988).

Immunoprophy laxis

Though substantial evidence is there for the presence

of functional protective immunity during hookworm infection,

but the molecular basis of the protective immune responses

has not been well defined. The strongest evidence for the

acquired immune responses to hookworm infection comes from

an early demonstration that the infective larvae of A.caninum

elicit immunity in dog (Herricke, 1928; McCoy, 1931; Foster,

1935). Different mechanisms have been documented for the

active evasion of the hos t ' s immune responses in many parasitic

10

infections and it is supposed that hookworms also employ similar

strategies for evading or suppressing the host protective res ­

ponses (Hotez et al_., 1987). The protective immunity to animal

hookworm parasi tes has been investigated in some detail (Miller,

1971; Carroll and Grove, 1984) and has been found to be asso­

ciated with both serological (Ball and Bartlett , 1969; Ogilvie

et a l . , 1978) and cellular responses (Ogilvie and Jones, 1973;

Taylor and Turton, 1976; Maxwell et_ a]_., 1987). However, a

strong correlation between these phenomena and host protective

immunity has yet to be conclusively establ ished.

Although, a number of investigations have been con­

ducted on the systemic immune responses of host to hookworm

infection (Kassai, 1982) and the possible role of local anti­

bodies in conferring immunity to the parasites (Poulain et_ a l . ,

1976; Sinski and Holmes, 1977, 1978; Wedrychowicz et a]_.,

1983a, b , 1984a,b), very l i t t le information is available about

the antigens important in the stimulation of intestinal antibody

responses. It has been suggested that IgA antibodies are in­

duced by antigens irrelevant to parasite survival and these

antibodies are supposed to play important role in establishing

host-parasi te relationship rather than evoking protective immunity

(Befus and Bienennstock, 1982). The most striking immunological

property of helminth infections is a capacity to provoke high

levels of IgE and reagin type antibodies in their hosts . Studies

concerning the function of IgE in parasit ic infections began

on the firm basis with the work on intestinal anaphylaxis

11

in N.brasiliensis infected rats (Urquhart et_ a]_., 1965; Jarrett

and Bazin, 1977). Both parasite specific and non-SDecifically

potentiated IgE responses have been reported during N.brasiliensis

infection m rats (Ogilvie, 1967; Orr and Blair, 1969; Bout

et a l . , 1977). However, subsequent studies established that

non-specific IgE does not protect against allergic reactions

as the passive transfer of immunity could be achieved using

antisera devoid of anaphylactic activity (Jones et_ al^., 1970;

Leid and Williams, 1974; Jarret t et_ a]_., 1980).

Various mechanisms have been proposed for the induction

of protective immunity in hosts; these are antibody mediated

immunity, cell-mediated immunity, and lastly the local inflamma­

tory responses (Jarret t and Urquhart, 1971; Ogilvie and Jones,

1973; Love et a l . , 1974). Immunity to mouse hookworm N.dubius

is believed to have two stages: antibodies damage the worms

which are then predisposed to antibody independent expulsion

from the gastrointestinal tract (Prowse et_ al^., 1978). This

self cure phenomenon seems to be achieved by a combination

of all the three mechanisms proposed earl ier for protective

immunity. However, passive transfer of protective immunity

to mice with immune sera against N.dubius infection, demons­

trated that serum antibody mediate protective immunity (Jones

and Robin, 1974; Dobson and Owen, 1978). Later on, it was

suggested that passive transfer of anti-N.dubius serum directly

or indirectly influenced the survival , growth and fecundity

of N.dubius (Dobson, 1982). It has also been found that the

12

pas s ive t r ans fe r of IgG, - fraction of anti-N .dubius «?erum

pro tec ted the mice and resu l t ed in the r e t a rded growth of

the p a r a s i t e s . When combined with adop t ive ly t rans fe r red

immune mesentr ic lymph node c e l l s , enhanced worm expulsion

from the in tes t ine was also obse rved ( P r i t c h a r d e^ a l . , )983).

According to the e a r l i e r r e p o r t s , i t was suggested that worms

p rev ious ly damaged by the host immune responses we>-p found

to be more suscep t ib le to expulsion mechanism as rompar-'-d

to the undamaged worms (Ofrilvie and Love, 1974). However,

in some c a s e s , the worms taken from the hos ts shor t ly before

expulsion and t r ansp lan ted to the o ther hos ts were no more

suscep t ib le to expuls ion (Hopkins et_ al^., 1972; Hopkins and

Zajac, 1976: Wakelin and Wilson, 1979). It has been suggested

tha t worm expulsion was due to the metabolic c r i s i s through

which the worms were going and the findings support the

en te roa l le rg ic ind i r ec t mechanism for worm reiect ion (Kassai

et a l , , 1987). Recent ly , some work has been done regarding

the expuls ion phenomenon of N . b r a s i l i e n s i s and free r ad i ca l s

(oxygen r a d i c a l ) a re sa id to be involved in the expulsion of

p r imary infections (Smith and Bryant , 1986; Smith, 1989).

Hookworm p a r a s i t e s have a lso been shown to evoke

cel l mediated immune responses (Ali et_ a^. , 1986). Infection

with A .duodenale has been found to be assoc ia ted with marked

cell mediated immunity as manifested by increased T-cel l counts,

increased inhib i t ion of leucocyte migration and increased lympho-

b las t t ransformat ion . An inve r se r e l a t i o n s h i p was proven between

13

the degree of cel l mediated immune responses and the oa ras i t e

load . Fu r the rmore , some o the r group of workers have also

r e p o r t e d the p r o b a b l e role of cel l mediated immunity m vacc i ­

nated an imals . According to t he se worke r s a delayed hyper

sens] t ]v i tv react ion has been obse rved m pups vaccinated witbi

i r r a d i a t e d infect ive l a rvae of A .caninum and t h i s skin reaction

has h<-f"ti reoor ted to cor re la te with th*- jnhibi t ion ot rhif msH'"*

tiop oi leucocytes in the presence of larvae or adult worm

antigen (Gautum et_ aJ^., 1986).

A successful induction of p ro t ec t i ve immunity with

soluble e x t r a c t s from adul t s of A.caninum and A.ceylanicum

has been shown by the reduction in worm burden bv 59% and

741 r e s p e c t i v e l y (Thorson, 1956; Car ro l l and Grove, 1984, 1986).

Dogs immunised with soluble e x t r a c t s p r e p a r e d from adult worms

have been found to be p a r t i a l l y r e s i s t an t to challenge infection

with L^ of A.ceylanicum (Car ro l l and Grove, 1985). Double

dose of i r r a d i a t e d L- l a rvae of A.caninum gave 90% protection

to subsequent chal lenge (Mi l le r , 1965, 1971). The route ol

adminis t ra t ion i s also an important f ac to r . in t raper i toneal

or subcutaneous route has been shown to be les^ effective as

compared to oral adminis t ra t ion which gave 100% reduction in

worm burden (GuDta and Kat iya r , 1985). Menon and Bhonale

(1985) have vaccinated the golden hamste r s with i r r a d i a t e d

larvcie of A.ceylanicum and obtained 95% reduction in worm

burden . However, such vaccines p r e p a r e d from i r r a d i a t e d

at tenuated l a rvae cannot be employed in human beings because

14

of some e th i c l e r e a sons , such as the danger of vaccinated host

becoming infec ted .

Characterization of antigens

Attempts to analyse the somatic e x t r a c t s from adult

and l a r v a l s tages of A.caninum and t h e i r c ross r e a c t i v i t v with

sera obtained from N.americanus infected pa t i en t s were made

long back by Williams (1970), using immunoelectrophoret ic t ech­

nique . The l a r v a l antigens could be sepa ra ted into 18 p rec ip i t in

bands whereas the adul t antigenic p repa ra t ion showed the p r e ­

sence of 13 bands with homologous immune r a b b i t serum. Only

one < ro^s r eac t ive component wa'? observod bftwron A.raniniim

and N.americanus employing pat ient s e r a , whereas the e x t r a c t s

from N.americanus adul t worms have been r epor t ed to show

four p r e c i p i t i n bands (Will iams, 1970). In contrast to these

f indings , Desowitz (1962) , employing Ouchterlony gel diffusion

t e s t has shown more r e l a t i o n s h i p between the same stage of

different spec i e s than between the di f ferent s tages of the same

s p e c i e s . Later on, a compara t ive ana lys i s of p ro te ins of

A.ceylanicum and A . b r a s i l i e n s i s was done employing disc gel

e l e c t r o p h o r e s i s and an over a l l s im i l a r i t y between the protein

pa t te rn of the two p a r a s i t e s was obse rved (Yoshida et^ a l . ,

1977), Antigens of 250, 60, 45 and 30 kD have been found

to be common between the adul t and l a r v a l s tages of N.dubius

(Hurlev et^ a]_. , 1980; Mitchell and Munaz, 1983; Mitchel] and

Cru i se , 1984; Monroy et^ £!.• » 1985; Monroy and Dobson, 1* 86;

Adams et a l . , 1987). Affinity pur i f ied fraction of adult worms

15

containing 55 and 20 kD antigen has been shown to induce oro

tection against infection with N.dubius (Monroy and Dobson,

1987). Charac te r iza t ion of antig-ens of N . b r a s i l i e n s i s adult

worm have also been done using monoclonal an t ibod ies (Bohn

and Konig, 1985). The antigenic pa t te rn of different d^veloo-

mental as well as the stage specif ic antigens of N.americanus

have been s tud ied using immune sera from human (natural host)

and hamste rs (Carr and P r i t c h a r d , 1987).

The antigens of nematode p a r a s i t e s have bc^n shown

to be excre ted or secre ted from the internaJ ce l lu la r and gJan

dular -structures during in v i t r o maintenance ot parasite->. Ih'=*se

e x c r e t o r y - s e c r e t o r y (L-S) antigens a re supposed to be Les"

heterogenous as compared to somatic e x t r a c t s and have been

found to play important ro le m diagnosis as well as m pro tec t ive

immunity. The role of F-S produc ts from hookworms m inducing

immunit ; *as repor ted by Otto and Kerr (1939j about a half

century ago. The e x c r e t o r y - s e c r e t o r y products of rat hookworm

pa ra s i t e N.muris have been shown to induce p ro t ec t i ve a n t i ­

bodies (Thorson, 1951). Later on, the p ro t ec t i ve role of

E-S products from a number of hookworm p a r a s i t e s was s tudied

by many workers and significant degree of protect ion was o b s e r ­

ved in p a r a s i t i s e d hos ts (Clegg and Smith, 1978; Iain et a l . ,

1979; Wedrychowicz et_ al_. , 1983). The immunogenicity of P-S

products from hookworm p a r a s i t e s was compared with that of

somatic antigens and it was suggested by many workers that

E-S produc ts were more immunogenic (Kat iyar et a l . , 1972 ; Goviia

16

et_ a]^. , 1973; Murray et_ al_., 1979). The E-S antigens of human

hookworm N.americanus have been analysed emplovine; immune-

blotting technique using serum from infected hamsters as well

as from the patients suffering from hookworm infection (Carr

and Pr i tchard, 1986, 1987). Ey (1988) has identified and charac­

terized the E-S products from exsheathed L- of Heligmosoides

polygyrus (N.dubius) and has also studied the role of E-S

products m protection. Besides, hookworms the E~S antigens

of other nematodes like T.canis (Sugane et_ al^., 1985), B.malayi

(Kaushal et_ al^., 1982, 1984), W.bancrofti (Maizels et al^., 1986),

S.cervi (Malhotra et_ al_., 1987) and Strongylus stercoralis(Brindlev

et al.,1988) have been successfully characterised using immuno-

electrophoretic and other immunochemical techniques.

The sex specific antigens present m the secretionh

of N.dubius adults have also been studied (Adams et aj^., 1988).

The E-S products of adult N.dubius have been shown to reduce

the ratio of male worms to female worms. A highly immuno­

genic molecule of 66 KD has been found to be present in the

secretions and also on the surface of males only and has been

supposed to induce protective immunity specific to male worms,

thus resulting in the less chances for mating and so the reduct­

ion in worm burden. The evidence for the cuticular contri­

bution to E-S products comes from an early demonstration that

N.americanus sheds significant amount of i ts surface antigen

into the medium during jji vitro incubation (Pritchard et a l . ,

1985). The adsorption of E-S antigens onto the surface has

17

been shown in case of T.spiral is (Silberstein and Despommier,

1985). Rats immunized with E-S products from Strongylus

stercoralis adults showed reduction in faecal ego; count as well

as adult worm burden (Mimori et_ al^., 1987), thereby suggestini?

the protective role of E-S products.

Surface antigens have also been shown to play a key

role in the induction and expression of host immune responses

(OgiJvie et ai^., 1980; Maizels et al^., 1982, 1983). The cuticle

of helminths including hookworms have been shown to be prone

to damage by antibody and complement and by many of the

antibody dependent cellular cytotoxicity reactions (Kazura and

Grove, 1978; Mackenzie et_ al_., 1978; Kazura and Aikawa, 1980;

Ogilvie et a l . , 1980). The stage specific surface antigen of

N.brasiliensis have been investigated using surface labelling

125 with 1, solubilisation with detergent followed by immuno-

precipitation and SDS-polyacrylamide gel electrophoretic analysis

(Maizels and Ogilvie, 1980; Ogilvie and Phi l l ip , 1980). The

cross reactions between the surface proteins of various nematodes,

which differ completely in their molecular weight characteris t ics ,

have also been documented by several investigators. Maizels

et a l . (1982) suggested that carbohydrate side chains could

be responsible for the cross-react ivi ty . The cuticular antigens

from N.americanus adults were studied by Pritchard et a l .

(1986), using sera from infected hamsters as well as human.

A 67 kD antigen of N.americanus has been reported to be pro­

duced in vitro also in measurable amounts (Carr and Pritchard,

18

1987) suggesting t h e r e b y the involvement of cut icular antigen

m inducing immune r e s p o n s e s . The surface antigens of

N.americanus have been shown to be h igh ly immunogenic and

have been found to be collagenous in nature which i s the mam

s t ruc tu ra l component of p a r a s i t e cut ic le (Keymer and Bundv,

1989). It has a lso been suggested recent ly bv P r i t cha rd et

a l . (1988a) tha t natural immune response against surface soeoi t ic

epitopp'- could be boosted with purif ied surface antigens TO

induro i sufficiently strong and p'^rsistr>nt r e s p o n s e s . It has

also been suggested tha t small collagenous molelcules and a s s o ­

ciated d i su l f ide bonds hold toge ther the cut ic le of N.americanus

( P r i t c h a r d et a l . , 1988b).

19

SCOPE AND PLAN OF WORK

Hookworm disease is a major public health problem

affecting millions of people and causing profound morbidity

and mortality. Chemotherapy has emerged as the only proce­

dure for temporary cure of the disease. However, repeated

doses of anthelmintic drugs are toxic and cause various types

of side effects. Therefore, the knowledge about molecular

immunology of hookworm infection is of urgent need as it

may provide certain leads for development of vaccines.

Several hookworm species are known to infect men

and l ivestocks. Due to the strong host specificity, Necator

americanus and Ancylostoma duodenale; the two human hookworms

are less amenable for the s tudies. Another hookworm parasite

Ancylostoma ceylanicum, (which infects man besides cats and

dogs), being maintained in golden hamsters (Mesocricetus auratus)

has extensively been used for chemotherapeutic and immuno­

logical s tudies. Many efforts have been made for studying

immune responses and protective notential of hookworm para­

s i tes , but the antigens responsible for evoking these r e s ­

ponses are not well defined. The characterisation of antigens

will provide the basic information not only for understanding

the host-parasi te relationship but also for identitying the

antigenic targets of protective immunity. Hence, in the present

study an endeavour has been made to analyse the somatic

and excretory-secretory antigens of A. ceylanicum. The present

dissertation deals with the following aspects:

20

I. Antigenic analysis of somatic extracts from adult and

infective larval stages of Ancylostoma ceylanicnm

1 . Protein pattern of adult and larval stages

of A.ceylanicum.

2. Immunisation of rabbi ts with somatic extracts and

quantitation of parasite specific antibodies in immune

sera .

3 . Antigenic analysis of adult and larval stages of

A.ceylanicum using immune rabbit sera .

4 Identification of common or cross reactive as well

as stage/parasite specific antigens of adult and larval

stages of A.ceylanicum.

5. Immunisation of hamsters with adult and larval somatic

extracts of A.ceylanicum and evaluation of immunised

hamsters for protective immunity.

II. Antigenic analysis of excretory-secretory products of adult

A.ceylanicum

1. Short term jji^ vi t ro maintenance of A.ceylanicum adult

worms and preparation of excretory-secretory products.

2. Immunisation of rabbi ts with excretory-secretory pro­

ducts and quantitation of parasite specific antibodies

in immune sera .

3. Antigenic analysis of excretory-secretory products

using immune rabbit sera .

21

4. Analysis of common or cross reactive antigens between

excretory-secretory products and somatic extracts

from adult and larval stages.

5. Immunisation of hamsters with excretory-secretory

products and evaluation of immunised hamsters for

protective immunity.

CHAPTER II

MATERIALS AND METHODS

22

PARASITES

Ancylostoma ceylanicum

Ancylostoma ceylanicum infection was or ig ina l ly procured

from Sarabha i Research Cent re , Baroda, Ind ia . The infection

was maintained in golden hamste r s (Mesocricetus aura tus) weigh­

ing 60-80 g by giving an inoculum of 60-65 infective l a rvae

( L - ) . Infected hams te r s were k i l l ed 21 days post infection

and the adul t worms were col lected from the mucous laver

of small i n t e s t i ne , p a r t i c u l a r l y the jejunal port ion by cutting

i t longi tudinal ly throughout i t s l eng th . The worms were then

washed thoroughly with isotonic sal ine containing ant ibiot ic

mixture (penic i l l in 100 U/ml + qtreptomycin 80 \)g/ml) to rprnovp

the adher ing in tes t ina l d e b r i s and bac t e r i a l contamination.

The clean worms were used immediately for antigenic p r epa ranon

or fcepT frozen a1 -20 C until u sed .

infect ive l a rvae of A. ceylanicum were collected trom

the coprocul tures of infected h a m s t e r s , 20-30 days post infect­

ion according to the method of Ray et_ al_. (1972). The faecal

pe l l e t s were emulsified with d i s t i l l e d water to a th ick pas te

which was then sp read over the centre of a moist Whatman

No.l f i l t e r p a p e r , kept in p e t r i d i s h e s and incubated in a humid

chamber at 27±2 C for about 5-6 d a y s . The l a rvae thus col lected

were suspended in d i s t i l l e d water containing an t ibo i t i c s and

washed thoroughly to remove extraneous mater ia l and the imour i -

t i e s of faecal ma t t e r . The clean l a rvae were then subjected

to mi l l ipore f i l t ra t ion (5 um) ui o r d e r to remiove the bacter ia l

23

contamination. The bacteria free larvae were recovered by

low speed centrifugation and kept frozen at -20 C until used.

Nipp>ostrongylus brasiliensis

Nippostrongylus brasi l iensis infection obtained from

Wellcome Laboratories of Tropical Medicine, London, were main­

tained in albino ra ts (UF strain, 25-35 g ) . Rats infected

with an inoculum of 2000 L- of N. brasi l iensis were kil led,

7-10 days post infection. The intestines were cut longitudinally

throughout the whole length and the clean adults , sticking

to the mucosal layer of jejunum, were obtained by baermanni-

sation of the suspension through muslm cloth. The adults

thus obtained were washed with lukewarm isotonic saline contain­

ing antibiotic mixture and stored frozen at -20 C until used.

Infective larvae of N.brasiliensis were collected essent­

ially according to the method of Keeling (1960). Coprocultures

were made from rats infected with N.brasi l iensis , 10-15 days

post infection. The paste of faecal pel le ts , made with isotonic

saline was mixed with granular charcoal in a ratio of 1:3.

The paste was then spread over the centre of moist filter

papers kept m petr i dishes and incubated at 27±2°C in a humid

chamber. The larvae developed after 4-5 days and migrated

towards the edge of the fil ter paper on attaining the infective

larval stage. The motile larvae were harvested by trimming

the edges of fil ter papers and baermannisation of the larval

suspension. The larvae were washed repeatedly with isotonic

14

sal ine containing an t ib io t ic mixture (pen ic i l l in 100 U/ml and

s t reptomycin 80 ug/ml) and s to red frozen at -20 C until used .

CHEMICALS USED

Acrylamide , aga rose , b i s a c r y l a m i d e , bovine serum

albumin, Coomassie Br i l l i an t Blue (R-250) , goat a n t i - r a b b i t

Ig p e r o x i d a s e and TEMED were procured from Sigma Chemical

Company , St . Louis , MO, U.S.A. The powdered media DMEM

and RPMI-1640 were obtained from Gibco Labora to r i e s , New

York, U.S.A. Ammonium pe r su l f a t e , beta mercaptoethanol were

t h e p roduc t s of E .Merck , A.G. Darmstadt , West Germany. F r eund ' s

Complete and Incomplete adjuvants were obtained from Difro,

Detro i t , MI, USA. All o the r chemicals used were of analvt ica]

grade and t r i p l e glass d i s t i l l e d water was used for a l l the

e x p e r i m e n t s .

PREPARATION OF MEDIA

Rosewell Park Memorial Institute 1640 (RPMI-1640):

The powdered medium supplemented with L-glutamine

(10.4 g) was d i s so lved in approx imate ly 900 ml of water and

then 5.94 g HEPES was added and the final volume was made

upto 960 ml . The medium was s t e r i l i s e d by f i l te r ing through

0.22 urn membrane f i l t e r s . P r i o r to use 4.0 ml of 5% s t e r i l e

sodium bicarbonate was added pe r 96 ml of medium.

Dullbeco's Minium Essential Medium (DMEM):

The powdered medium with E a r l e ' s sa l t and L-glutamine

was d i s so lved in approx ima te lv 900 ml of double d i s t i l l ed

25

water. Sodium bicarbonate (3.7 g) was then added and the

volume of the solution was made upto one l i t r e . The medium

was steril ized as described for RPMI-1640.

Ringer's Solution:

Weighed quantities of sodium chloride (9 g) , potassium

chloride (0.42 g) , glucose (0.25 g) and Ca.n]2 (0.42 g) were

dissolved in a minimum amount of water. After raising the

volume upto 900 ml, separately dissolved Tris (1.21 g) was

added and the volume was made upto one l i t r e . This was

then steri l ised as described for RPMI-1640.

PREPARATION OF ANTIGENS

Somatic antigens

Soluble antigenic extracts from adult and larval stages

of A.ceylanicum and N.brasil iensis were prepared essentially

according to the method described by Kaushal et al_. (1982)

for the preparation of filarial antigens. Briefly, lyophilised

adults/infective larvae were ground to fine powder in a pestle

and mortar and suspended in isotonic saline (0.15 M NaCl).

The suspension was sonicated 4 times for 1 min each with

an interval of 1 min using an ultrasonic cell disruptor (Heat

systems, Ultrasonics Inc. Ltd. NYW-220 F) at 20 Kc/sec, and

extracted on ice for 4-5 h, with continuous gentle st irr ing.

The extracts were then centrifuged at 10,000xg for 30 min

and the supernatants thus obtained were used as a source of

26

somatic antigens. These antigenic extracts were kept frozen

at -20 C in small aliquots until used.

Excretory-secretory antigens

Fresh, motile worms obtained from the infected hamsters

were thoroughly washed with isotonic steri le saline containing

a mixture of antibiotics (penicillin and streptomycin). Separate

batches of 100 adult worms were incubated at 37 C in 20 ml

of three different media (Ringer's solution, DMEM and RPMI-

1640) containing penicillin (100 U/ml) and streptomycin (80

ug/ml). The media collected at the end of incubation period

were lyophil ised, dialysed and used as a source of excretory

secretory antigens.

Immune rabbit sera

Polyvalent hyperimmune sera were raised in albino

rabbi ts (weighing 1.0-1.5 kg) against somatic extracts prepared

from adult and larval stages of A.ceylanicum and N.brasi l iensis .

Rabbits were immunised intramuscularly with an emulsion of

somatic extracts (1 mg protein) made in Freund's complete

adjuvant. One month after the first injection, three subsequent

injections were given at one week interval and the rabbits

were bled from the marginal ear vein, one week after the

fourth injection. After tha t , immunisation and bleeding of

the rabbi ts was done alternatively at weekly intervals t i l l

the hyperimmune serum was obtained. The collected blood

was allowed to clot by keeping at 37°C for 1 h and then left

overnight at 4 C. The serum was collected by centrifuging

at 800xg (10 min) and stored at -20 C in small aliquots.

27

Polyvalent hyperimmune serum afi;ainst e x c r e t o r y - s e c r e t o r v

p roduc ts from A.ceylanicum adu l t s was a lso r a i s ed in r a b b i t s

( 1 . 0 - 1 . 5 kg) by giving 0.5 mg prote in emulsified in Freund' s

complete ad juvant . The immunisation and bleeding schedule

followed was same as d e s c r i b e d for somatic an t igens .

For ra i s ing an t ibod ies against normal hamster serum,

r a b b i t ' were immunised with 1.0 mg prote in ot normal hamster

>eruni eraiusified with Freund ' s complete ddiuvanx: a?- dfescrit-ed

lor somatic an t igens . The serum obta ined was s tored in small

a i iquots at -20 C until used .

PREPARATION OF GAMMA GLOBULIN FRACTION

Gamma globulin fraction from immune r abb i t sera was

p r e p a r e d by adding ammonium sulfate (45% sa tura t ion) to the

serum with continuous gentle s t i r r i n g at room tempera ture for

30 min and then overn ight at 4 C. The p r e c i p i t a t e obtained

was recovered by centrifugation at 10,000xg for 30 min and

d i s so lved in minimum amount of isotonic s a l i n e . The ammonium

sulfate was removed by d ia lys ing against t h r e e changes of

isotonic s a l i ne . The gamma globulin fraction was centrifuged

at 800xg for 15 min and s tored at -20 C in small a i i q u o t s .

IMMUNISATION OF HAMSTERS

Golden hamste rs (Mesocricetus a u r a t u s ) , weighing M~

80 g were immunised with somatic e x t r a c t s (100 ug prote in)

ot A«^.^_l£nicum adult and l a rvae and E-S products (SO ug orutein)

ot A.ceylanicum adul t p a r a s i t e emulsif ied with F r e u n d ' s complete

28

ad iuvan t . The immunisation was c a r r i e d out by giving a total

of five injections subcutaneously at 15 days i n t e r v a l s . Hamsters

were b led one week af ter each injection and the sera collected

were s to red at -20 C unti l u sed . One week after the 5th in ­

jection the hams te r s were challenB;ed with 60 L^. The animals

were k i l l e d 21 days post chal lenge and the worm burden was

recorded and compared to unimmunised infected con t ro l s .

ENZYME LINKED IMMUNOSORBENT ASSAY

Enzyme l inked immunosorbent assav (ELISA) was p e r ­

formed according to VoUer et_ al_. (1974) . Br ie f ly , 100 ul

(0.015 ug - 1.0 ug) of A.ceylanicum adult somat ic / l a rva l somatic/

adult e x c r e t o r y - s e c r e t o r y antigens d i lu ted in phospha te buffered

sal ine (0.01 M, pH 7.2) were added to each well of a mic ro-

t i t r e ELISA p la te and incubated for 1 h at 37 C and then at

4°C ove rn igh t . The p la t e was washed with PBS-Tween ( p h o s ­

pha te buffered sal ine containing 0.05% Tween-20) to remove

the unbound ant igen. Two hundred mic ro l i t e r s of 1% BSA (made

in O.OIM PBS, pH 7.2) were added to the wel ls and the p la te

was incubated for 1 h at 37 C in o r d e r to block the uncoated

s i t e s . The p la t e was washed t h r i c e with PBS-Tween. Different

d i lu t ions of serum (100 ul) were then added to the wells and

p la te was kept at 37 C for 2 h . After washing t h r e e t imes

with PBS-Tween the p la te was incubated at 37 C for 1 h with

100 ul of pe rox idase l abe l l ed conjugates of goat a n t i - r a b b i t

Ig ( 1: 1000) / rabbi t an t i -hams te r Ig (1 :500, p r epa red in our

l a b ) . After t u r t h e r washing 100 ul of subs t r a t e solution

29

(1 mg/ml 0-phenylene diamine in c i t r a t e buffer 0.05 M, pH

5 .2 , containing 1 u l /ml of hydrogen pe rox ide ) was added to

each we l l . The react ion was s topped af ter 10 min with 50

ul of 5N H^SO. and the r e s u l t s were r eco rded as extinction

at 492 nm. The amount of colour change (OD) wil l be p r o p o r ­

t ional to the amount of ant ibody in the t e s t serum.

SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS

SDS-polyacrylamide gel e l e c t r o p h o r e s i s (SDS-PAGE)

of A.ceylanicum adul t and l a r v a l somatic e x t r a c t s was performed

according to the method of Laemmli (1970) using Bio-rad s lab

gel e Jec t rophores i s appa ra tus {Bio-rad Labora to r i e s , U . S . A . ; .

A separa t ing gel of 10-20% acry lamide concentration was made

with the he lp of a gradient m i x e r . The composition of 10%

acry lamide gel mixture was : 5 ml of s tock acry lamide (30%):

b i s ac ry lamide (0.8%) mix tu re , 3.75 ml of 1.5 M Tris-HCl

(pH 8 . 8 ) , 0.15 ml of 10% SDS, 0.05 ml of 10% ammonium per

su l fa te , 0.005 ml of t e t r ame thy le thy lene diamine (TEMED) in

the to ta l volume of 15 ml . The 20% acrv lamide gel mixture

contained 10 ml of s tock a c r y l a m i d e : b i s ac ry lamide and 1.8

ml of g l y c e r o l . The r e s t of the components were added to

the mixture in t h e same amount as d e s c r i b e d above for 10%

ac ry lamide gel m i x t u r e . The volume was made upto 15 ml

wxth d i s t i l l e d w a t e r . The s tacking gel (3%) contained 1.0

ml of ac ry lamide stock solution (30% a c r y l a m i d e : 1.0% bis acry la ­

m i d e ) , 2.5 mi of 0.5 M Tris-HC] (pH 6 . 8 ) , 0.1 mi of 10% SDS,

2.5 ml of 60% suc rose , 0.05 ml of 10% ammonium per sulfa te ,

30

0.005 ml of TEMED in a to ta l volume of 10 ml. The samples

were p r e p a r e d by mixing equal volume of the antigen ex t r ac t s

(100-150 ug/20 ul) with SDS sample buffer (containing 10 ml

g lyce ro l , 5 ml of be t a -mercap toe thano l , 23 ml of 10% SDS,

1 ml ot 0.1% bromophenol b lue , 8.32 ml of 0.5 M Tris HCl,

pH b . 8 , in a to ta l volume of 50 ml) in 1:1 ra t io and keeping

for 5 min in a boil ing water b a t h .

E l ec t rophore s i s was c a r r i e d out at a constant current

of 25 mA per gel for about 4-5 h t i l l the t racking dye reached

to 1 cm above to the bottom of the ge l . After e l e c t r o p h o r e s i s

the gel was s ta ined with 0.25% Coomassie Br i l l iant Blue (R-

250; made in 50% methanol p lus 10% acet ic ac id) and desta ined

with 30% methanol p lus 7% acet ic a c i d .

IMMUNOELECTROPHORESIS

Immunoelectrophoresis ( lEP) was performed according

to the method of Graber and Williams (1953) . The somatic

e x t r a c t s and E-S p roduc t s from A.ceylanicum were separa ted

e l e c t r o p h o r e t i c a l l y in 1.5 mm th i ck l a y e r of 1% (w/v) agarose

made m b a r b i t a l buffer (20.0 mM d ie thy l b a r b i t u r i c ac id ,

80.0 rriM T r i s , 2.0 mM sodium a z i d e , 0.8 mM calrmm lact^ti",

pH 8.b( toy i n at 100 v o l t s . After eiectrophoresi-^ nne aidT«<-.

were Kept over night in a moist chamber and the separa ted

antigens were allowed to react with immune r abb i t s e r a . The

p la tes were then washed with isotonic sal ine for 30 min and

p r e s s e d under s eve ra l shee t s of f i l t e r paper for 10 min. This

p rocedure of a l t e rna te washing and p ress ing was repea ted t h r i ce

31

and the p l a t e s were washed with d i s t i l l e d water before the

final p r e s s i n g . After a i r d ry ing the p l a t e s were s tained with

0.25% (w/v ) Coomassie Br i l l ian t Blue (R-250) made in 45% ethanol

p lus 10% acet ic acid and des ta ined with t h e same.

CROSSED IMMUNOELECTROPHORESIS

Crossed immunoelect rophores is (CIE) was performed

according to the method of Axelsen et_ al_. (1973). The antigens

were f i r s t s epa ra t ed e l e c t r o p h o r e t i c a l l y in the f i r s t dimension

m 1.5 mm t h i c k l a y e r of 1% (w /v ) agarose at 10 V/cm. A longi­

tudinal s t r i p (1x7 cm) containing t h e sepa ra ted antigens was

cut and t r a n s f e r r e d to t h e another p l a t e (7x9 cm) . Anodic

(5x7x0.12 cm) and ca thodic (2x7x0.12 cm) gels containing immune

serum (5-20%) were then poured on the p l a t e . An in termediate

gel s t r i p of 1x7x0.13 cm without an t ibody was also poured

m between t h e f i r s t and t h e second dimension g e l s . The an t i ­

gens were then al lowed to move e l e c t r o p h o r e t i c a l l y m second

dimension for 18-20 h at r i gh t angle to the f i r s t dimension

gel (2-3 V/cm) . After e l e c t r o p h o r e s i s the p la tes were washed

and s ta ined in t h e usual way as d e s c r i b e d for lEP.

CROSSED LINE IMMUNOELECTROPHORESIS

Crossed l ine immunoelectrophoresis (CLIE) is the modi­

fication of CIE and was c a r r i e d out in a manner s imi lar to

tha t of CIE except tha t m CLIE the t e s t antigen (protein :

50-100 ug) was added m the in te rmedia te ge l . After the e lect ro­

ph o re s i s the p l a t e s were washed and s ta ined in a way s imi lar

32

to that of lEP. On staininf?, the antigenic peaks which give

lines at their bases are considered to be common between the

two antigen ext rac ts .

TANDEM CROSSED IMMUNOELECTROPHORESIS (TCIE)

This is the modification of crossed immunoelectrophoresis

in which the two antigen samples were mixed together and

placed in one application hole. The electrophoresis was carried

out in the same way as for CIE. The common antigens from

the two samples fuse to give an increase in the heights of

the peaks. The control plate contained only the standard

antigen. The washing and staining procedures were exactly

the same as described earl ier for lEP.

PROTEIN ESTIMATION

Protein contents of the somatic extracts from adult

and infective larvae and excretory-secretory products were

estimated according to the colorimetric method of Lowry et

a l . (1951).

CHAPTER III

RESULTS AND DISCUSSION

SECTION I

ANTIGENIC ANALYSIS OF SOMATIC EXTRACTS

FROM ADULT AND INFECTIVE LARVAL STAGES

OF ANCYLOSTOMA CEYLANICUM

33

The somatic antigens from the adult and larval stages

of hookworm parasi tes are capable of inducing various types

of serological reactions but the antigens involved in these

responses have not been well defined. Characterization of

parasite antigens is of particular significance not only for

understanding the host parasite relationship but also for identi­

fying the immunologically important components of the parasi tes.

Due to the differences in the habitat and nutritional require­

ments, the protein and antigenic compositions of different

parasites as well as of different developmental stages of a

parasite may be different. Therefore, a basic knowledge

about the total protein and antigenic make up of different

stages of a parasite is a necessary pre-requisi te and may

provide useful leads about the antigens having role in pro­

tective immunity. Accordingly, in the present study, detailed

investigations were carried out to analyse the protein and

antigenic mosaic of adult and infective larval stages of

A. ceylanicum employing certain biochemical and immunochemical

techniques.

PROTEIN PATTERNS OF ADULT AND LARVAL STAGES OF

A. CEYLANICUM

Somatic extracts from adult and infective larvae of

A. ceylanicum were analysed on SDS-PAGE and their protein

patterns are shown in Fig. 2. The molecular weight of various

proteins present in these extracts was determined by running

xn paralleJ the standard proteins of known molecular weights.

34-

A B MW

94 67

43-

30

r~5

r-10

[—IS

i -20

i -25

— 35

f—AO

i—45

'—50

J/k*"'

r -10

'r-15

20

i—25 30

— 35

40

i—45

•—50

Fig. 2: SDS-Polvacrylamide gel electrophoretic pattern of adult

(A) and infective larvae (B) of A. ceylanicum. The

protein bands in both adult and larval stages are num­

bered according to their movement on the gel and bands

having similar electrophoretic mobilities are given

the same numbers. All the bands are marked on the

figure irrespective of their presence or absence in

a particular stage.

35

The protein bands were numbered according to their electro-

phoretic mobilities and the bands having identical electro-

phoretic mobilities in both the stages were given the same

number (Table 1 ) . Fourty seven protein bands in Ac A and

43 protein bands in AcL were observed in the molecular

weight range of 10-170 kD. Most of the protein bands detected

in L_ were also present in adult somatic extracts with minor

differences in thei r intensit ies, however, 8 protein bands

specific to adults and 4 bands specific to larvae were observed.

ANTIGENIC ANALYSIS OF ADULT AND LARVAL STAGES OF

A.CEYLAmCUM

( i ) Quaotitation of parasite specific antibodies in immune

rabbit sera by enzyme linked, immunosorbent assay

The antibody t i t e r s of immune rabbit sera raised against

the somatic extracts from adult and larval stages were deter­

mined by enzyme linked immunosorbent assay (ELISA). The

assay system was standardised in terms of optimum concentration

of antigen, antibody-conjugate, o-phenylene diamine (OPD)

and hydrogen peroxide (H_0_). Out of different dilutions

of goat ant i-rabbit immunoglobulin-peroxidase conjugate tested,

1:1000 dilution showed optimum resul ts . This dilution of

the antibody conjugate was used in further experiments. Different

concentrations of OPD and H_0_ were also t r ied and 1 mg/ml

of OPD and 1 ul/ml of H^O was found to give optimum ELISA

specific ac t iv i ty . The optimum concentrations of AcA and

36

Table 1: Comparat ive ana lys i s of p ro te ins of aduJt and in­

fect ive l a rvae of A. ceylanicum.

Protein band number

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

Apparent molecular mass (kD)

170.0

160.0

153.0

145.0

140.0

130.0

125.0

120.0

100.0

94.0

88.0

85.0

80.0

73.0

66.0

64.0

60.0

55.0

47.0

45.0

43.0

42.0

40.0

39.0

38.0

37.0

36.0

35.0

34.0

33.0

32.0

31.0

Adult

-

-

-

++

+ + +

++

+

+

++

++

+ + +

-

+

+ + +

++

+ + 4

+

+ + +

+ +

+ + +

+ +

+ +

+

+

+ +

+ +

+ +

±

+

+

+ +

+ +

Larvae

+

++

+

+

++++

-

-

+

+

+

+ + +

+

+

++

++

++

++

+

+

+

+

-

++++

+++

+++

+

+

+

+

+

+

+

( c o n t d . . . . ]

37

33 30.0

34 29.0

35 27.0

36 26.0

37 25.0

38 22.0

39 20.0

40 19.0

41 18.0

42 17.5

43 17.0

44 16.5

45 16.0

46 15.0

47 14.0

48 13.0

49 12.0

50 U.O

51 10.0

+

+

+ +

++

++

+

+

++

+

+++

+

+++

+++

+

++

+++

+

+

+

—

±

+

+

±

+

-

++

+

+

-

-

+++

±

++

+

+

+

_

The adul t and infect ive l a rvae of A.ceylanicum were solubi l ized

in 1 X SDS-sample buffer (containing 5 ml g lyce ro l , 2.5 ml

^ -mercap toe thano l , 2.5% SDS, 0.001% bromophenol b lue , 0.0625M

Tr i s HCl, pH 6 . 8 , in a to ta l volume of 50 ml) by keeping

in a boil ing water ba th for 5 min and sepa ra ted on 10-20%

SDS-polyacrylamide gradient g e l . The prote in bands were num­

bered according to t h e i r molecular w e i g h t s .

++++ = ve ry in tense , +++ = in tense , ++ - modera te , + = weak,

± = faint

38

AcLwere determined by using different concentration of ant i­

gens (0.015 to 1 ug/well) at fixed dilutions of immune rabbit

serum (1:500, 1:1000, 1:2000) and it was found to be 0.25

ug/well for Ac A and 0.50 ug/well for AcL. No significant

increase in ELISA specific activity was observed by increasing

the antigen concentration upto 1 ug/well, however, on decreasing

the antigen concentration, decrease in ELISA specific activity

<ff'As noticed (Big. 3,4) .

After optimising the assay system, the antibody t i ters

of parasite specific antibodies in immune rabbit sera were

determined and the titration curves are shown in Fig. 5 and

6. The titration curves obtained with AcA using homologous

(rabbit anti-AcA) and heterologous (rabbit anti-AcL) immune

rabbit sera showed linearity upto a serum dilution of 1/32,000

(Fig. 5) . The reciprocal antibody t i t e r s of 256,000 and 128,000

were obtained with homolgous and heterologous sera respect­

ively . The titration curves obtained with AcL using rabbit

anti-AcL (homologous) and rabbi t anti-AcA (heterologous) sera

are shown in Fig. 6. Both the titration curves were linear

upto the serum dilution of 1/32,000 and a reciprocal antibody

t i t e r of 256,000 was obtained with both homologous and hetero­

logous immune rabbi t sera .

( i i ) Antigenic pattern

Immunoelectrophoretic analysis: Antigenic characterization

of the somatic extracts was initially done bv immunoeie-ctro

39

^

0.20 O.AO 0.60 0.80

Ant igen concentrat ion ( / j q / w e l l )

• • 1

1 0

Fig. 3: Effect of different concentrations of adult somatic extract of A.ceylanicum on the specific activity in enzyme linked immunosorbent assay using 1:500 (X-X), 1:1000 (0-0) and 1:2000 (£»-A ) dilutions of rabbi t , anti-A.ceylanicum adult serum.

40

0.20 O.AO 0.60

Antigen concentration ( / jg /we l l )

0.80 1

1.0

Fig. 4: Effect of different concentrations of larval somatic extract of A.ceylancium on the specific activity in enzyme linked immunosorbent assay using 1:500 (X-X), 1:1000 (0-0) and 1:2000 (A—A ) dilutions of rabbit anti-A.ceylanicum larval serum.

41

o o o

•\ o o . T —

o o o

o o o

c o 3

•6 E 3

o -7^

{J

0 9.

cr

rt •1-'

0 «

0 0 >^ - H

nl ^ H •<-'

' ':i •H 3 •^ "TJ C (TJ

rt "

* 1 •rt a J3 0 n t 18

•

?5 rt w 0) rt

4) • M ; « - ^

3 0 0) 73 -5 JD

1 ^ 0

•M « 0

rt 0 D

*- 5 1 - « "

sh X

^ V C 0 +J -H '-' -H 1—1

(a

C «l 0)

(4 B •^ 0 c

(Z6!^QQj/^jiAjpe oijiDads

i n

«c

42

ir>

CM

J^^

^ ^ v o

^. 6 • " •

( Z 6 ' 7 Q O ) ^iiAnop Diioadg

I • in

c3

O If o o

o o o o ^

o o o

c o 3

E 3 61 in

« • -

o _ <0 u o a u

cr

(B •(-> u u 0) nl en u

•t->

X o <" 1

••->

n)

rt 0 > to

- i «) 3

73 Sr 5 o

^ >-

X 2 (0

^ - ^

•H -H -H ^ y

c

to 0) c «i E 0 1 s,

nl ^ C u a

•

b4

43

phoresis ( lEP). The antigenic extracts from adult and larval

stages were separated electrophoretically and then allowed

to react with hyperimmune rabbit sera . Ten tn twelve preci­

pitin lines were observed in AcA with rabbit anti-AcA serum,

while antigenic preparation from infective larvae showed the

presence of only 6-7 precipitin bands with rabbit anti-larval

serum (Fig. 7 ) .

Crossed immunoelcctrophoretic analysis (CIE): Antigenic com­

ponents of adult and larval stages of A.ceylanicum were further

separated more clearly using two dimensional Immunoelectro­

phoresis , i . e . crossed Immunoelectrophoresis (CIE). Different

concentrations of serum (gamma-globulin fraction) were tr ied

in order to get bet ter resolution of antigenic components.

Antigenic peaks became much stronger and sharper with increas­

ing the antibody concentration, however, with higher per­

centage of serum, slight decrease in the height of antigenic

peaks was observed which may be due to shift in the zone

of equivalence.

When somatic antigenic preparation from adult worms

(AcA) was allowed to react with i t s homolgous serum (20%-

gamma-globulin fraction of rabbit anti-adult serum), 34 antigenic

peaks could be observed, 25 moving towards the anode and

9 towards the cathode. Fig. 8a shows the protein stained

CIE pattern of AcA while i ts diagrammatic representation is

shown in Fig. 8b. Each peak represents a single antigen

and the faint antigens are shown by the broken l ines. Almost

4-4-

Fig. 7: Immunoelectrophoretic patterns of somatic extracts from

adult and infective larval stages of A.ceylanicum adult

somatic extract (A) and larval somatic extract (B).

1 = Rabbit antiadult serum; 2 = Rabbit anti-larval

serum.

4-5'

Fig. 8: Crossed immunoelectrophoretic pattern of A.ceylanicum

adult somatic extract antigen (a) Protein stained pattern,

(b) Diagrammatic representation. Solid lines represent

strong antigenic peaks . Arrows indicate the point

of application of antigen. Anode ( + ) is to the right

in the first dimension and to the top in second dimen­

sion gel. Antibodies: 20% gamma-globulin fraction

of antiserum raised against somatic extracts from adult

A. ceylanicum.

46

similar antigenic pattern was observed using 15% and 20% gamma­

globulin fraction, however, with a lower antibody concentration

of 10%, 3-4 antigenic peaks (numbering 5,7,11 and 15) d i s ­

appeared completely and the intensity of some of the antigenic

peaks declined. Reduction in the number of cathodic preci ­

pitin peaks was also observed with 10% serum concentration.

When larval antigenic preparation (AcL) was run with

rabbit anti-AcL serum (10% gamma globulin fraction), 19 anti-f

genie peaks could be observed. The antigenic pattern of

the somatic extracts from AcL, as revealed by CIE, i s depicted

in Fig. 9. Out of 19 antigenic peaks 17 were anodic and

2 were cathodic. One of the two cathodic antigens (AcL-

18) was slow migrating and precipitated in the first dimension

gel only. Using a lower concentration of gamma-globulin fraction

(5%), some of the faint peaks numbering 1, 3 and 11 d i s ­

appeared completely and a decrease in the intensity of some

of the antigenic peaks (numbering 4 and 8) was also observed.

Table 2 summarises the results obtained by lEP and

CIE analysis of AcA and AcL using the homologous rabbit

sera.

IDENTIFICATION OF COMMON OR CROSS-REACTIVE AND STAGE/

PARASITE SPECIFIC ANTIGENS

Antigenic similarit ies do exist among different stages

of a parasite as well as between two different paras i tes ,

hence studies were also conducted to identify the common

Fig. 9: Crossed immunoelectrophoretic pattern of A.ceylanicum

larval scrmtic extract (a) Protein stained pattern, (b) Dia­

grammatic representation. Solid lines represent strong

antigenic peaks . Arrows indicate the point of appl i ­

cation of antigen. Anode ( + ) is to the right in the

first dimension and to the top in second dimension

gel. Antibodies: 10% gamma-globulin fraction of anti­

serum raised against somatic extracts from larval

A.ceylanicum.

48

• n

H u

(0 0) bf rt

+ j en

1—1

«! > yi

1—1

T( C rt

•»-> r-H 3

T) «)

M H

O

(0 •H tn >-• — 1

rt c rt o

• H

c t) bf

• H 4 J

c <

• • N

4) F H

Xi J^ H

E 3 U i) (n

•H

c <

c i) b£

•H +-> C <

m 0) 3 O

• H C

Si u d)

H

c rt •H 4)

O 73 O

•rt U5

C <

< u <

I 1 • H ••J c rt

-4-> • H J2 J3 A

Oii

J U < 1 1

•H •fJ

c (t

+-> • H J3 X) (6

K

< <

u <

1 o u • » ->

o a;

f-H

0) o c 3 E E

)—i

w • H w (U H

o XI

a

in r-CO r-H

< < I

•H c

J3

u <

I

c (0

•H XI Si nt

OS

<

I

u <

0 05 C -H 3 m E Ji 1 o

to i o "

U 0)

• » - »

u nl U

+ j

X (U

u • H + J

rt E o (0

+ J r-H

3 TJ

O nt U

• • - »

X (U

u • H

•*-> nl E 0 n

r—(

n! > rt rt r^

B 9 U

1 •< ft)

u

B 9 U •H B

43 •< ft)

u <l <l

< o <

49

or cross-reactive antigens between the adult and larval stages

of A.ceylanicum and also between the two hookworm parasites

(A.ceylanicum and N.bras i l iensis) .

(i) Common/cross-reactive and stage specific antigens

The common/cross-reactive antigens between the adult

(AcA) and larval stages (AcL) of A.ceylanicum were analysed

by doing CIE of AcA and AcL using immune rabbit sera raised

against these stages. When the somatic extracts from adult

worms were allowed to react with rabbit anti-AcL serum in

CIE, 22 antigenic peaks were observed (Fig. 10b) while CIE

analysis of A.ceylanicum larval antigen (AcL) with rabbit

anti-AcA serum showed the presence of 14 antigens (F ig . l ib ) .

On comparing these results with CIE patterns of AcA (Fig.8)

and AcL (Fig. 9) with the homologous immune rabbit sera,

12 and 5 antigens appeared to be specific to adult and larval

stages respectively (Table 3) .

The presence of common or cross-reactive and stage-

specific antigens between adult and larval stages was further

confirmed by doing cross-line Immunoelectrophoresis (CLIE).

Fig. 10c shows the CLIE pattern of adult antigen in which

the larval antigen was introduced in the intermediate gel

s t r i p . On comparing the CLIE pattern of AcA (Fig.10c) with

i ts CIE pattern (Fig. 10a), most of the adult antigens appeared

to be common to the larval stage as indicated by the presence

of lines at the bases of these antigens. However, the antigens

STO

n) U

c •H 01 D

O A

-t-> X

E o CO

3

u o

a o o 4}

1—I 4) O C D E E

a; c

t) in (0 o

0) ••->

ei -

c u

•» ->

+-•

a

0)

o x: a o o i)

<u o c 3 E E

E •3

>

c

Xi

in )H

t3 c

(U E u 9)

J3

3

O O

o n)

+->

o a 0)

o o +->

J3

nj o T3 c

o u

<

E 3 U en

3

« (

c w

C rt

c o

•iH U) c <u E

-H

en

be

ifl flj

Si +-> H

c o

o

X 0)

0) E o en

E 3 U 0)

en

3 J2 O

•—I «

E E rtj 61)

T3 O

^H be O c

o c

<

c 4)

+-> c

c o

c 0 in

•H c t) E

(A in 0 u

3

(0 I H

•» ->

c (TJ

c (U

•(-J

m

a

3

bC

a a (0

-a c o u u (A

s

51

> (0

T3 c n!

nt

w c 4) bt •H c

o

0 -

be

c

u n) (U ^ I tn en O ) H

o c o E 6 o o

A)

E2

B 9 U

•H

o

c u

< l

o

o tn

- tt) a (A

be

<U C bc n) n)

I W CO O *H

u B O E E o u

c 0) bf •H •< ->

B (Tt

0) >

•H • U U n) (1)

m •H

(U ^ E 0) 1^ br

3 CT

• H

c

o

H

E

3

CO

c <

to B (U b f

•H

c <

u •r-l

O X! ••->

rt U

73 O c <

o • 4 ->

n) U

O B <

• in

* t ^

*

V

i n 00 00

u <

< u <

1 o

T3 1

1 O

T3 1

J O

<

< I <

w 1—t

o M •—1

u

u t—4

b j

u M 1—1

u u t—(

o

w •—1

hJ u

< u <

1 1 • H •t->

B rt

+ j • H

J3 J3 (0 «

J u <

1 1 • H •<->

B rt

••-> • H

^ J3 n)

CH

< o

< 1 1

• H + J

B rt

••-' •H

x> J3 (« «

J u <

1 1 •H 4 J

B rt

4-1 • H

X) ^ nt «

< o < 1 1

•rt •» ->

B rt

•4-> • H

XI XI n) «

J o < 1 1

•H •«-> B (d

+ j • H

-D XI n)

OS

I I 0 o 1 I

U

m

a)

nt E o CO

3 T3 (0

(J n)

+-> X

E o en

>

s 9 u •H

a ^ >> 0)

u

B 9 U •a 43 • 0) u

<l <l

< u <

tn E <l) bc

• H + J B

o

a to

3

< •Il­

ea

o x; a o

•t->

o

to (U u o x: 0-o u

• 4 - "

o 0)

I—( 0) o B 3

to E (1) br H

B

1—1

0 B 3

E E

E E

•fH

1 T3 ID 0) 0)

U C

a to 0)

CO CO

o u

CO CO

O O U

W w

o u

52

AcA 2, 3, 18, 19, 21, 24, 25 and 30-34 which did not have

lines at the bases appeared to be specific to adult stage.

CLIE of AcL was done by adding AcA in the intermediate

gel s t r i p . On comparing the CLIE (Fig. l i e ) with CIE (Fig.

11a) patterns of AcL, most of the larval antigens were found

common to the adult stage, however, 5 antigens (AcL-2,13,

16-18) specific to AcL were also observed. The results of

stage specific as well as common/cross-reactive antigens of

the two stages are summarised in Table 3.

( i i ) Common/cross-reactive and parasite specific antigens

A.ceylanicum antigens having cross-reactivity with

the rat hookworm parasite N.brasil iensis were also analysed

using heterologous immune rabbit sera (raised against adult

and infective larval somatic extracts of N.bras i l iensis) . CIE

of AcA with rabbi t anti-NbA serum revealed the presence

of 12 cross-reactive antigens between the adult stages of

the two parasites (Fig. 12a). When the AcA antigen was

allowed to react with rabbit anti-NbL serum, 8 cross-reactive

antigens between the two stages of both the parasites were

found (Fig. 12b). Common/cross-reactive as well as parasite

specific antigens of AcA were further analysed using CLIE.

Fig. 13a shows the CLIE pattern of AcA with NbA in the

intermediate gel s t r i p . On comparing the CIE pattern (Fig.8)

of AcA with i ts CLIE pattern (Fig. 13a), 22 antigens (AcA

1-3, 6, 9, 10, 12-14, 18, 19, 21, 25-34) were found to be

AcA specific. Fig. 13b shows the CLIE pattern of AcA using

o

: ^

+

o

\

\. + + . >

1—1

ti > u n)

1—1 •H • « ->

c

• H J3 Xi

B •H CO D

O A $ 1

i)

a •fH • ! - >

nj E o (fl

>-H

n) > ti

a 3 U

•H 0

>> 4) u • <

O

c u 4}

•t->

(0 a u

u o

a o u o 0)

r—1 0) o c 3 6 E

<4H

O

c u

a u

- H

(U u o

Si a o u

•*->

u 0)

1—I i) o c 3 E E

• f H

c •iH 1—I

•T3 4) (A (0 o

u

, ^^ Xi

E 3 ^H

V «

<—( 3

73 rt 1

•iH

c <fl

XI

c nJ

E 3 ^4

0)

ClC c • H (A 3

1-H

bt

0) • • J n)

• H

E

c • H

0)

-t->

c

tac 3

o o 1—1

^^ • ! - >

u n)

(J •rH • 4 - "

E 0 'n

•>->

1—1

3

• H

5

u >•«'

o R) tH

• ( - >

X

u • J

m E o (0

1—1

> (fl

1 nt u • H

1—1

a 0. n)

VtH

o

c • H

a • » ->

0) + J

rt o • H 73 c • H

tn ^ o

<

E 3

0)

1—1

n3 > ^H

<« 1

•r-t

c «J

-t-" M

Xi XI

<4H

0

c 0 H

+ J

o

c .—1

3 Xi 0

E E bc

c*> O

1 c 4) E

•H

XI

73 c 0 O 0) V)

c

a O

• 1 - '

u Xi - t - l

o •»->

TJ c

c o iH CO

c 0)

E • i H

TD

i H •-t-t

c • H

••->

be

(U XI

o

tn H

-f

0) T I O C <

• c i) be H

c n)

V M

o c 0 rt

c o

5

m + \h) +

n— H- -f-

Fig. 12: Crossed immunoelectrophoretic pattern of A.ceylanicum

adult somatic extract using 15% gamma globulin fraction

from rabbit anti-N.brasil iensis adult serum (a) and

rabbi t anti-N.brasil iensis larval serum ( b ) . Arrows

indicate the point of application of antigen. Anode

( + ) is to the right in first dimension and to the top

in second dimension.

es-

Fig. 13: Crossed line immunoelectrophoretic pattern of A.ceylanicum

adult somatic extract with N.brasil iensis adult somatic

antigen (a) and N.brasiliensis larval somatic antigen

(b) (100 ug of each in the intermediate gel) using

20% gamma globulin fraction of rabbit anit-AcA serum.

Arrows indicate the point of application of antigen.

Anode ( + ) is to the right in first dimension and to

the top in second dimension.

56

NbL in the in te rmedia te gel s t r i p . Only 8 antigens were

found common between AcA and NbL. Antigens numbering 1-

3 , 10-25, 27, 29-34 were found to be AcA spec i f i c . Table

4 d e p i c t s t h e summary of t he se r e s u l t s .

Common/cross- react ive antigens between the l a r v a l

s tages of A.ceylanicum and N . b r a s i l i e n s i s were a lso ana lysed .

CIE ana lys i s of AcL with r a b b i t ant i-NbL and r a b b i t a n t l -

NbA sera r evea l ed 13 antigens ( F i g . 1 4 a , b ) . On comparing

t he se r e s u l t s wi th CIE pa t t e rn of AcL with homologous ( r a b b i t

ant i -AcL) serum, 6 antigens appea red to be AcL spec i f i c . The

speci f ic and common antigens were fu r the r analysed by doing

CLIE. On comparing CIE ( F i g . 9) and CLIE (F ig . 15a) of

AcL, 13 antigens appea red to be cross- reac t ive /common between

the two p a r a s i t e s whi le antigens numbering 12-14, 16, 17 and

19 were found to be AcL s p e c i f i c . CLIE pa t t e rn of AcL with

NbA i s shown in F i g . 15b. On comparing the CLIE (F ig . 15b) ,

of AcL with i t s CIE (F ig . 9) most of the antigens were found

common between AcL and NbA. Antigens numbering 2, 13,14,

16, 17 and 19 appea red to be AcL s p e c i f i c . The r e s u l t s of

common or c r o s s - r e a c t i v e as well a s speci f ic antigens of AcL

as compared to NbL and NbA are summarised in Table 5 .

IMMUNIZATION OF HAMSTERS WITH SOMATIC EXTRACTS FROM

ADULT AND LARVAL STAGES AND EVALUATION OF IMMUNISED

HAMSTERS FOR PROTECTIVE IMMUNITY

Hamsters were immunised with the somatic e x t r a c t s

from adul t and l a r v a l s tages of A.ceylanicum at 15 d a y s i n t e rva l .

57

73 to U

a E o u

(fl n)

D T! («

B s u

• H

I 0)

u

(0

c 0) be

•H c nt

u (1)

a (0

' 0 c n)

0)

> • H o n) 0) v< I (0 (n o »^ u

c o B E o o

E2

(0 •r-l (0 c 0)

in n)

U

•t! = « "J

! a,

c I 4) to tiT

o (fl

E X2

o ™

•H

(U

(U 3 D"

• H c o t)

H

E D

0) tfi

c <

c i) bC

• H •t-> c

<;

o •r-( T l O

•4->

(fl

o

(J •H TJ O C <

T3 0

J3

(fl O

c <

(?

I r-l

fM

IT) (M

(M

Z z

w • — t

u u l-H

u

w • — 1

>- o

w h-l

u

u l-H I - ]

u

< u <

1 1 •H • f j

c <fl

4 J • H XI XI (fl ec

< X3 z

1 1 •H • f j

c (fl

••-» • H XI X) (fl «

< u <

1 1 •H + J

c (fl

•*-• • H X) XI (fl oi

J X) z

1 1

+ J

c (fl

•»-> •1 -4

XI X3 (fl «

< o <

1 I • H -tJ

c (fl

•IJ • H XI X5 (fl

Otf

u «fl

•t->

X

(fl

E 0 n

u (fl

X

(fl E o 0)

T3 rt

o (fl

•<-»

X 0)

(fl E o

(fl > Vi (fl

< o <

1 o

XI

1 o

13 1

1

o T) 1

1 O

n 1

(0

B s u •H

1 ^ 4) U

(» •H tn c 0)

•rt rH •H (0 ? )H XI

to •H tn c 0)

• H l-H •H tfi (fl V* XI

< l z l

< < o XI

< z

tn •rt (fl o; 0

x: 0-o V4 u V

4) 0 c 3 E E

tn 0

(0 4) ; H

o X! a 0 u

•(->

u 4)

l-H 4) 0 c D E E

• r t

4) C

X) 4J

tn o

• J M • - ( XI S J z o o

^

ta)

i s

- H- (bJ >

4-

iiiMHMi

a 4-

Fig. 14: Crossed immunoelectrophoretic pattern of A. cey lanicum

larval somatic extract using 10% gamma globulin fraction

of rabbi t anti-N.brasil iensis larval serum (a) and rabbit

( b ) . Arrows indicate

Anode ( + ) is to

anti-N.brasil iensis adult serum

the point of application of antigen.

the right in first dimension and to the top in second

dimension.

53

Fig. 15: Crossed line immunoelectrophoretic pattern of A.ceylanicum

larval somatic extract with N.brasil iensis larval antigen

(a) and N.brasil iensis adult antigen (b) (100 ug of

each in the intermediate gel) using 10% gamma-globulin

fraction of rabbi t anti-AcL serum. Arrows indicate

the point of application of antigen. Anode ( + ) is to

the right in f irst dimension and to the top in second

dimension.

60

(fi c 0)

•H .—I •H 01

JO

Z l

u 0) a tfl

OJ •M • H m

a,

-^ (U >

• H

•»-> u ni (L ^ 1 (fi tn 0

U

V) c V

• H -t->

c «)

w c t) bf

• H •t->

c rt c 0 E E o o

Tl 0 x: U

u •H T3 O C <

u • H T3 0

XI -t-"

n! U

o • H

1 c <

I r-l

•H Xi m r-l E l i

0)

3 cr

•H C

XI u d)

H

E 3 0) (0

c <

c 0) be

• H

c

< o

<

fVJ (N3

T3 < XI X I

I Z I 2

w 1—1

o u t—1

u

w 1—1

l-J u

u 1—1

u

w t-H

h j u

J u < 1 • H

+-" c rt

•<-» •rt -Q X3 rt a;

J XI 2 1 • H + J

c nJ

4-" • H

X) XJ rt

OJ

J o < 1 1

+ j

c rt

•»-> •rt ^ X3 m o;

< XI 2 1

• H • M c rt

+ j • H

X3 X5 n) «

J u < 1

• H 4->

c rt ^ • H

X3 XI n)

OS

1 o

T l 1

1 O

T3 1

1 0

T l 1

1 O

TJ

u

0)

E o m

>

o

<

o Rl u

•» ->

X 0)

rt E o (0

2

u n)

4-> X 0)

u •H

n) E o m

3

t-~t

B 9 U

•H

1 >> 0) u

m • H (0 c V

• H

• H

{* >H

XI

to • H (0 c 01

• H r-( •H

(fl ^ X!

21 21

o X3 0.

o ^4

-t-" o

I—I dj o c 3 E E

t 3 (U (A 0)

o ( J

< XI 2

tn •H to <D U O x: a o u

+ J o lU

i-H <u o c 3 E E 0) c

•V

tn in o u u

II

w W '-:!

O u

61

The sera obtained one week after every injection were tested

in enzyme linked immunosorbent assay (ELISA) and immuno­

diffusion (ID). The antibody t i te rs of immune hamster sera,

expressed as geometrical mean reciprocal t i ter (GMRT) are

given in Table 6. The parasite specific antibody could not

be detected in sera obtained after 1st injection, however,

significant levels of antibodies were detected in sera from

2nd injection onwards. The GMRT values obtained for hamster

anti-AcA and hamster anti-AcL serum after the 2nd injection

were 65.97 and 57.48 respect ively. The GMRT values of

these sera were significantly increased to 527.80 for hamster

anti-AcA and 303.14 for hamster anti-AcL following the 4th

injection. No significant change m the antibody t i ters of

these sera was observed after 5th injection. The immuno­

diffusion analysis of these immune hamster sera showed that

the precipitin bands first appeared after the 3rd injection

and 3 precipitin bands in AcA and 2 precipitin bands in AcL

were observed. The number of precipitin bands was increased

to 4 and 3 with AcA and AcL respectively after the fourth

injection and it remained constant after 5th injection. The

results of ID are summarised in Table 7. The hyperimmune

hamster anti-AcA and hamster anti-AcL sera obtained after

the 5th injection were tested in lEP, which showed the p re ­

sence of 7 and 4 precipitin bands in AcA and AcL respectively

(Fig. 16).

The hyperimmune hamsters were evaluated for protective

immunity by challenging them with an oral dose of 60 L-

01 (0

c 0)

U

o (0 o c D E E

c •H

E > N c 0)

JD

rt u (U (0

; (U

w E «J

V c a E E

(0

0)

t3 O

• H

c <

E2

c o

^ • ^ LTl 4)

• * - >

>-O

•H

c <

C O

c

C

o

E

m •H c <

c (V M

• H • ( - >

c <

00 (M

o

00

00

rj<

r-N i n

en o en

ID

O IT)

i n o

00

r~ o

• i n >o

00 •«i<

• r i n

Q Z

Q 2

u < I

c nl

U (U

• » - >

E <«

• J u < I

•H +J

c

U <u (A E n)

X

< <

+ J

o nJ

X (U

o •H + J

rt E 0 «

• < - '

r—1 3

B s u -g ^

u < II

< u <

• f j <J nt 4-> X 0)

u • f t 4-> n) E o (0

»—1 nl

> nl

•—1

5 a u •H

>>

<

II

J o <

(0

(U ••-> (D

E nl

X

i> >

•H M-l

E 0 u

t) C

•H

rt

0

H

o

a> "•J • H ••->

1—t

nl u O U & u

c nJ

E .—( nl U

• H

0) E o C)G

(fl nl

T3 0)

0)

a X ' CJ t-J

o CJ <

nl 73 c

en n)

^ < •H O

>> U T ) O

o '^ :2x -t-> u C nl

< i)

0)

o 0) +-> (U

T3

T3 O

•H +-> c nl

o 2

II

Q 2

62

63

C

m 3

S s

H u

rt » •(->

X (1)

o • H - t J

<B

E 0 m

r—(

> u ta

r—<

T) c rt

• * j

3 73 nl

>+-i

0

to • H

(0

>-r - H

n) c rt

c o •H Ul D

• H

T) 0 C D

E E

• rt >-( 0) m

u i)

••-> OT

E

(U c 3

E E

E2

Cfi Ti C rt

XI

j :3 •(->

>

c o •H o <u 'f~

a

o tt)

a

o

>

•H

c o

^ .2.

c o

c

c o

tn -^

tn •H +-> c <

c i) be

<

E 3 U 0) (0

< o <

1 • H 4->

c (6

y, 4)

•»->

CD

E n a:

< u <

E 3

^ 0) cn

J u < 1 1

+-> c m u <D

•(-> CO

E rt ac

.-) o <

c o

o

o

4)

a)

4) C O

T3

(U

& (A

tt) +J to E

X!

tt)

H

•V 4J N

•H c 3 E E

•H

U CO

tt)

CO E rt

Xi

O

rt X

X o rt tt)

c tt) E

•rt tt) a X 4)

tt) tt)

JC

c rt • tt) cn

e g rt c

tt) ^H o tt) g

C tt) tt) J :

•H

O CO tt) j 3 3 <j rt

tt) rt > 4) ^

X -H H &

B a u t

^ •> 0 u

B s u •H

a

V u 1

<l

u a u •M X tt)

(0 E o CO

3

rt

<

<l

I tt)

rt E o CO

> U

o

^4-

A

Fig. 16: Immunoelectrophoretic patterns of somatic extracts from

adult and infective larval stages of A.ceylanicum using

. immune hamster serum. A = adult somatic extract;

B = larval somatic extract . 1 = Hamster anti-adult

serum; 2 = Hamster anti-larval serum.

65

S 9

E o u

o re ;H

X 0)

o

re >

re

T3 c re

3

re

0

c <u o a

> •H +-> o (U o

00

1)

E2

c 0)

c o

o

^ c

0) !H U 3

<U a.

T3 0) c

XI O (D

be m n p <u r '-I J-i - — I o re ^ X !

o o u

E D 2

C 0) txp

•H -M C

<

CO O O I

o o

o o I

CO CO

I

i n +1 o +1

i n +1

(U c o

TJ c re

05 u 0)

E re

x :

< u <

o <

0) X

re

^ -o w

re + j • ^ w

• H

-0.5 S E

13 re

• 1 - '

•—{

3 m 73 Jj re >

m <^ 0 J

o _ ^ c •H . •

O <u

o + T3 C

tie ^ re 3 . ,

o c ? 0 0 ) + ^ .—1 r—1

re j - i X X :g • M O

!* <U 0 X

T) -^ x : <u _ <J to 5 "1

•H O (U

g o x

- H

a , i n .N ^ c

^ •a 0) c

to + j c >H ^ E <a '^ -1-^

-t->

jn ^ 0) E <u >H re <u 0)

•X. ^ ^

+-> o re

X

(J

re E o

r—1

3 T) re

B 9 U

•H B (0

i-H

> V u • <

II

< u <

+ - i

o re u

4->

0)

(J • H

re E o Vi

re >

re 1—1

B 9 U

-3 ^ •> V u • <

II

J o <

en - M

c 0)

E • H

1)

a X 0) 0) 0)

X

<4H

0

c re 0) E

<u X -f-»

0)

^ re

0) 3

.—1

re > 0)

X H

66

at one week after the 5th injection. The worm burden was

seen on autopsy at 21 days post challenge and percent reduction

in worm burden gave the degree protection. The hamsters

immunised with adult somatic extracts gave 63% reduction m

worm burden while 93% reduction in worm burden was observed

in hamsters immunised with larval somatic extracts (Table

8 ) .

DISCUSSION

Ancylostoma ceylanicum (a human hookworm parasite

maintained in golden hamsters) has extensively been used

for chemotherapeutic and immunological studies by several

workers (Misra et_ al_., 1981; Katiyar et al^., 1982; 1984; Kamath

et al^., 1985; Carroll and Grove, 1985; Gupta and Katiyar

1985). However, no information is available about i ts total

antigenic composition as well as about the antigenic molecules

involved in inducing protective immune responses. In the

present study an effort has been made for analysing qualita­

tively , the protein and antigenic make up of adult and infective

larval stages of A. ceylanicum employing SDS-polyacrylamide

gel electrophoretic, crossed immunoelectrophoretic and crossed

line immunoelectrophoretic techniques. The levels of parasite

specific antibodies in sera raised against the two life stages

(adult and larvae) of A. ceylanicum have been determined

67

employing enzyrfle linked immunosorbent assay technique. The

protective potential of these somatic extracts has also been

evaluated. Since both male and female worms have been

shown to be equally effective in conferring protective immunity

(Gupta and Katiyar, 1985), the antigenic preparations made

from both female and male worms has been used in the present

study.

The protein composition of adult and larval stages

of A.ceylanicum was studied by SDS-PAGE, which provides

a high resolution of protein bands according to their molecular

weight and the best separation of proteins was obtained using

10-20% gradient of acrylamide gel. Lower percentage of acryl-

amide in the gel showed poor resolution as most of the pro­

teins present in these stages were of low molecular weight

proteins. This technique also enables a comparison of protein

patterns of different parasites preparations on the same gel.

SDS-PAGE has earl ier been used for studying the protein

patterns of many nematode parasi tes v iz . Ascaris (Zayatas,

1978), D.viteae (Neilson, 1978; Prusse et_ al^., 1982; Lucius

et a]^., 1983); hookworm (Wange et_ al^., 1980), Trichinella

(Bany et_ al_., 1976; Efremove and Ermolin, 1981); Angiostrongylus

cantonensis (Kum and Ko, 1985) and S.cervi (Malhotra et

a]^., 1986). In the present study, SDS-PAGE helped in the

separation of 47 and 43 protein bands in adult and larval

stages respect ively, of which larval stage exhibited only

13 major protein bands as compared to 28 observed in adult

68

stage. These results revealed a less complex nature of larval

somatic ex t rac ts . The different life stages of some other

nematode parasi tes have also been shown to have approxi­

mately the similar number of protein bands (Kaushal et a l . ,

1982; Prusse et a l . , 1982; Malhotra et_ al_., 1986). However,

earl ier studies of Yoshida e^ al^. (1977) have shown the p re ­

sence of only 13-15 protein bands in A.brasiliensis and

A.ceylanicum adults by disc gel electrophoretic analysis .

In the present study SDS-PAGE analysis revealed

some interesting qualitative and quantitative differences between

the two stages of A.ceylanicum. Protein bands numbering

1, 2, 3 and 12 (170, 160, 153 and 85 kD) were found to be

specific to the larval stage while protein bands numbering

6, 7, 22, 33, 39, 43, 42 and 51 (130, 125, 42, 30, 20, 17,

16.5 and 10 kD) appeared to be adult specific, as they were

only present in the larvae and adults of A.ceylanicum. A

comparison of stage specific proteins indicated that most

of the adult specific proteins (except protein bands 6 and

7) were below 50 kD, while 3 out of 4 larvae specific proteins

were above 150 kD. Carr and Pri tchard (1987) have also

shown low molecular weight (19 kD) protein specific to

N.americanus adult . A 74 kD protein specific to larvae of

N.americanus was also observed by the same workers .

In the present study, the levels of parasite specific

antibodies in immune rabbit sera were determined by enzyme

linked immunosorbent assay (ELISA) which has extensively

69

been used for quantitation of antibodies against a number

of helminth parasi tes including hookworms (Oeilvie et_ a l . ,

1978; Tandon et_ al^., 1981, 1983; Novoselska et_ aJ . , 1981;

Wedrychowicz et al^., 1984; East et_ al_., 1988; Roach et_ a l . ,

1988;Ey, 1988; Malhotra, 1988). The assay conditions were

optimised in order to get the optimum resul t s . The per­

oxidase labelled goat ant i-rabbit Ig conjugate was used to

measure the total antibody responses in the immune rabbit

sera . The rabbit anti-adult and rabbit anti-larval serum

showed high t i te rs of parasite specific antibodies against

both adult and larval somatic antigens thereby showing a

high level of cross-react ivi ty between the two stages of

A.ceylanicum. This technique has been used successfully

to detect the antibody levels against N.brasiliensis(Wedrychowicz

et ad., 1984) and N.dubius (East et_ al^., 1988). ELISA has

also been used to detect the cross reactivity between the

two helminth parasi tes T.trichuria and T.muris (Roach et

al^., 1988).

Immunoelectrophoretic techniques have generally been

used to study the antigenic compositions of several parasites

and have also proved to be useful in the identification of

common or cross-reactive as well as stage and parasite specific

antigens (Williams, 1970; Neilson, 1978; Kaushal et_ al_., 1982;

Lobos and Weiss, 1985; Cavalan et_ a]_., 1985; Malhotra et

al^., 1986). In the present study the techniques of immuno-

electrophoresis (lEP) and crossed immunoelectrophoresis (CIE)

70

have been employed to analyse the antigenic patterns of adult

and infective larval stage of A.ceylanicum. The antigenic

analysis by lEP revealed less number of precipitin bands

in the somatic extracts from the larval stage of th is parasite

while the somatic extracts from the adult stage showed more

precipitin bands. On the contrary, Williams (1970) has shown

more complex pattern for infective larval stage of A. caninum

than for adults using the same technique of Immunoelectro­

phores is . This technique has also been used to analyse

the antigens of some cestodes v iz . Hymenolepis nana and

H.diminuta and showed the presence of 14 and 9 precipitin

arcs with their respective sera (Manas et_ al_., 1985). Employ­

ing the same technique of lEP the antigens of several helminth

parasi tes and also the cross-reactive antigens among these

have been analysed (Choi and Lee, 1979).

Antigenic characterisation by CIE also revealed lower

number of antigens in the infective larval stage as compared

to adults of A.ceylanicum. This technique helped in better

separation of antigens from these stages. Various concentrations

of gamma-globulin fraction from hyperimmune rabbit sera

were tried in order to get a sharp and balanced pattern

of the antigenic peaks and a 20% concentration of gamma­

globulin fraction was found to be optimum for resolving the

antigenic pattern of adults of A.ceylanicum, while a 10%

roncf^ntration of gamma-globulin fraction was required

for the separation of larval antigens. On lowering the anti-

71

serum concentration further, the faint antigenic peaks could

not be seen clearly while the higher concentrations resulted

in poor resolution of some of the closely placed antigenic

peaks. The CIE technique showed the presence of 34 antigens

in adults and 19 in larvae of A.ceylanicum. Out of the two

cathodic antigens of infective larvae of A. ceylanicum, one

was slow migrating and precipitated in the first dimension

gel only. This could be due to the presence of a low charge

on the antigen. CIE technique has also been used for antigenic

analysis of many helminth parasites including hookworm para­

s i t e s . Twenty six antigenic components have been visualised

by CIE in the extracts of N.brasil iensis (Cavalan et a l . ,

1985). Kaushal et al - (1982) have demonstrated the presence

of 35 antigens in the extracts from B.malayi adults using

the hyperimmune rabbit serum. At least 21 antigens in the

isolates of 0.volvulus (Lobos and Weiss, 1985) and 22-24

antigens in S.cervi adults (Malhotra et_ a![,, 1986) could be

detected employing the same technique of CIE. This technique

has also been used to bring out similarit ies and subtle diff­

erences in some other parasi tes v iz . D.viteae (Neilson, 1975),

Ascaris (Kornishina, 1977), S.cervi (Malhotra et al^., 1986).

Protective immunity to hookworm infection can be

induced by immunisation with specific antigens (Pritchard

et a l . , 1984). Cross protection has earl ier been reported

for many helminth parasi tes (Au and Ku, 1979; Ericksen,

1982; Lee et a l . , 1982) and the cross-reactive antigens of

72

certain helminth paras i tes , like tapeworms, whipworms have

been identified (Milner et_ ail_., 1987; Correa-Oliveira et a l . ,

1988; Roach et al_., 1988). Cross protection has also been

observed between A.ceylanicum and N.brasiliensis (Gupta

and Katiyar, 1985). In view of these findings an analysis

of the common or cross-reactive as well as specific antigens

between different stages and also between different parasites

would be important for understanding the mechanisms involved

in protective immune responses. In the present study we

have analysed the common/cross-reactive as well as stage

specific antigens displayed by two developmental stages of

A.ceylanicum. Common or shared antigens exist between the

two stages as evident by the reactivity of somatic extracts

from these stages with heterologous sera in CIE as well as

by CLIE analaysis. CIE analysis of AcA and AcL with sera

against both these stages (adult and larvae) showed the p re ­

sence of 12 (AcA) antigens specific to adult and 5 (AcL)

antigens specific to larval stage. CLIE analysis also revealed

same number of stage specific (12 adult specific and 5 larvae

specific) antigens of A.ceylanicum. The presence of common

antigens between the adult and the larval stages of A.caninum

has been reported earl ier employing immunoelectrophoretic

technique (Williams, 1970). Common or cross-reactive antigens

between different stages as well as stage specific antigens

have been identified for other parasi tes including hookworms

viz . D.viteae (Neilson, 1978); B.malayi (Kaushal et aJ_.,

73

1982); T.spi ra l is (Phil l ip et_ al^., 1980; Parkhouse and Clark,

1983; Parkhouse and Ortegga, 1984); S.cervi (Malhotra et

a l . , 1986; Kaushal £t_ al^., 1987); N.americanus (Carr and

Pr i tchard, 1987); N.dubius (Adams et_ aJ^., 1988); P.knowlesi

(Singh, 1990).

In the present study an effort has also been made

to analyse the common or cross-reactive antigens between

the two hookworm parasi tes ; A.ceylanicum and N.brasi l iensis .

Antigenic cross-reactivi ty between the adult and larval stages

of A.ceylanicum and N.brasil iensis was studied using CIE

and CLIE. These studies showed that out of a total of 34

antigens present in A.ceylanicum adul ts , only 12 and 8 were

found to be common -or cross reactive respectively with adult

(NbA) and larval (NbL) stages of N.brasi l iensis . However,

the larval stage of A.ceylanicum showed 13 (out of 19) common

with both adult and larvae of N.brasi l iensis . These studies

have also revealed A.ceylanicum parasite specific antigens.

Presence of cross-react ivi ty among different helminth parasites

has been shown ear l ier and the cross reactive/common as

well as parasite specific antigens have been identified (Williams

1970; Rao et_ al^., 1980; Turner e; al^., 1980; Maizels et aJ^.,

1983; Gottstein et a l . , 1986; Kennedy et_ al^., 1987; Roach

et_ a]_., 1988).

Most of the studies conducted so far have shown

that protective immunity to hookworm parasi tes can be induced

In experimental animals by live parasi t ic stages and HJSO

74

by immunising with somatic extracts from adults and larvae

(Foster, 1935; Miller, 1971, 1979; Carroll and Grove, 1985;

Gupta and Katiyar, 1985). In the present study the protective

potential of A.ceylanicum antigens has been evaluated by

immunising the hamsters with somatic ext rac ts . The sera

obtained from the hamsters immunised with AcA and AcL,

before challenge, showed significant levels of parasite specific

antibodies. The immunodiffusion and lEP analysis revealed

the presence of precipitin antibodies in these immune sera

as well as the number of AcA and AcL antigens recognised

by these sera . The immunised hamsters when challenged

with live infective larvae showed significant resistence to

challenge infection. The infective larval stage proved to

be better in inducing protective immunity as it exhibited

over 90% protection (reduction in worm burden) as compared

to 63% obtained in the hamsters immunised with adult stage.

Carroll and Grove (1985) have observed 74% reduction in

worm burden in dogs immunised with the somatic extracts

from adult A.ceylanicum About 76% reduction in worm burden

was obtained in hamsters immunised with live infective larvae

of A.ceylanicum (Gupta and Katiyar, 1985).

Therefore, in the present study we have been able

to obtain basic information about the protein and antigenic

composition of adult and larval stages of A.ceylanicum.

These studies have revealed less complex nature of infective

larvae as compared to the adults as evident by the less

75

number of proteins and antigens in the larval stage. Certain

common or cross-reactive antigens between the two different

stages (adult and larvae) of A.ceylanicum and between the

two hookworm parasi tes (A.ceylanicum and N.brasilieisns)

have also been identified. These studies also showed the

presence of certain stage and parasite specific antigens of

A.ceylanicum as indicated by CLIE, analysis . The level

of cross reactivity between the two life stages has also been

studied by quantitating the parasite specific antibodies in

immune rabbit sera by ELISA. On evaluating the protective

potential of adult and larval somatic extracts , it has been

found that the larval stage is capable of exhibiting high

degree of protection, however, i t certainly needs further

evaluation in order to determine the possible mechanism of

protective immunity and also to identify the antigenic molecules

involved in inducing the protective immune responses.

SECTION II

EXCRETORY-SECRETORY PRODUCTS FROM

ADULT ANCYLOSTOMA CEYLANICUM

76

The excretory-secretory (E-S) products, released

during HI vi tro incubation of live paras i tes , are known to

be less complex in nature than somatic antigens and have

been shown to be of potential importance not only for establish­

ing specific diagnostic techniques (Maizels et al^., 1984, 1986;

Brindley et al^., 1988; Marrero et al^., 1988) but also in induc­

ing protective immunity against certain helminth parasites

(Thorson, 1951; Jenkins and Wakelin, 1983; Sugane and Oshima,

1983; Maizels et al^.. 1984; Gamble, 1985; Urban and Romanowski,

1985; Silberstein and Despommier, 1985; Badley et ad., 1987;

Mimori et a l . , 1987; Ey, 1988). Although a few reports are

available showing the protective potential of E-S products

released by the hookworm parasi tes (Katiyar et al^., 1972;

Govila et_al_., 1973; Murray et al^., 1979; Ey, 1988a,b), l i t t le

is known about the antigens present in these products. This

may be due to the difficulties in obtaining sufficient quantities

of E-S products . In the present studies we have optimised

the conditions for the preparation of E-S products from the

adults of Ancylostoma ceylanicum and analysed them employing

certain immunoelectrophoretic techniques. The protective

potential of E-S antigens was also studied.

PREPARATION OF E-S PRODUCTS

The conditions for the preparation of E-S products

from adult A.ceylanicum parasites were standardised. Different

media (RPMI-1640, DMEM and Ringer's solution) were tried

77

for short term j ^ vitro maintenance of A.ceylanicum adults

and the quantities of E-S products obtained in different media

are shown in Table 9. Among the three media used. Ringer's

solution proved to be the best for the preparation of E-S

products as the motility of worms was found to be maximum

in th is medium and the worms could be maintained up to

6 h without the change of medium. Analysis of E-S products

showed the release of 125±25 ug protein per 100 adult worms

in Ringer' s solution, while the protein released in the other

two media (RPMI-1640 and DMEM) was 95±15 ug per 100 adult

worms. Immunoelectrophoretic analysis of the E-S products

prepared in different media, using immune rabbit serum raised

against adult soma.tic antigen (AcA), also revealed almost

the same number of precipitin bands in all the three E>S

preparations i . e . 5-6 precipitin bands in RPMI-1640 and DMEM

and 6-7 in the Ringer's solution (Table 9 ) . Hence, in further

experiments, the preparation of E-S products was done using

Ringer's solution. In order to determine the optimum time

for the collection of E-S products, the adult worms were

maintained iji vi t ro and the E-S products were collected at

different hours by changing the medium at 2 h intervals .

The maximum amount of protein (125 ug/100 adult worms)

was released by the worms during the initial two hours.

After that , a decrease in the amount of protein released was

observed and i t became one fifth during 6-8 h period of

in vitro incubation (Table 1 0 ) . ^ ^ came sluggish

Ace No,

78

0) (0

(1) I—I

s

B 9 u

4) U

u a

T( O U

a

o 4-" i) u u t) <J) I

!>. o

4-> (U VH

u X w

Xi

E2

13 0) E

c U i)

(U

E 3

z

a, w

0) C

D-J3

c +J O c o o o

T3 S bc 4J ? 3

(0

E o

C -•H (0 <U rt •!->

E 3

•H

<u

+1

in

o

3 +J •rt +J (0

c

u o E 0)

(0 OH

(A O

I I

+1

E 3

•H TJ

<1)

c <u (A 10

w E 3 E

o o 0)

J3

in CM +1 in

C O

O

i)

c 0) E

• H

^ (U P-X (U

+J c 0) v< 4)

MH •H tJ

4) 4)

S H 0

c m 4} E 4}

J3 •*->

v >H nj

en + j 1—(

3

4)

4J XI H

CU W 1—1

c • H

4) 05 3

03 (fl ^

E 3 V, 4) Oi

< O < • H •» ->

c a

XI J3 (0 «

79

Tl 0}

• M

o (U 1—1 1—<

0 u +-> r—I 3

T) rt

B 9 U

•H C

^ >> u u •

< 4-1 o

o

T) 0 ^H

a

0 V

1 >-u o

u o

• (fl r—(

rt > >H

ii •»-> c •H

3 0

xi 0 ^

+ J

•t->

c o H

p .—1 s (0

0)

c

.a

OS

en T) c

XI

S o-o.

E « 3 >H _

Z D-.S

E O

c 0)

+J O 0) c o o o

'^ ft C (U l>

•H to

IT I i n I.

i n

+1 in (M

+1 o in

+1 o in

in +1 in (NJ

(NJ

O 1 1

(VI

1 1

0 0

1

(U

>

u 4)

c o

o tn

<u bf c

•H 06

E 3

• H

- O 4}

E

(n

0)

0) O

•(->

c

0} •4-1 (0 c (\)

(U

u

(fl E o

tn

3 O

O

80

after 6 h of incubation and could not regain their motility

even after transferring them to the fresh medium. Hence,

an incubation period of 6 h was used further for the collection

of E-S products. Fig. 17 depicts the lEP analysis of E-S

products collected at every two hours. The number of bands

was maximum ( i . e . 6-7 precipitin bands) during the initial

two hours and the number gradually decreased to 4-5 in the

E-S products collected during 2-4 h and 4-6 h of incubation.

Only 3-4 precipitin bands could be observed in the medium

collected during 6-8 h of incubation.

IMMUNISATION OF RABBITS AND QUANTITATION OF PARASITE

SPECIFIC ANTIBODY BT ENZTME LINKED IMMUNOSORBENT ASSAY

The rabbi ts were immunized with E-S products (pre­

pared in Ringer's solution) and sera obtained were tested

for parasite specific antibodies by enzyme linked immunosorbent

(ELJISA). The assay conditions such as the optimum concent­

rations of OPD, H-0_ and optimum dilution of antibody conju­

gate have already been described in Section I. In order

to determine the optimum antigen concentration different concent­

rations of E-S antigens (0.015 ug to 1 ug/well) were tested

at three fixed serum dilutions of rabbit anti-E-S serum (1:500,

1:1000, 1:2000) and it was found to be 0.50 ug/well. After

that , there was not significant increase in ELISA specific

activity on increasing the antigen concentration to 1 ug/well

(Fig. 18). After determining the optimum concentration of

81

Fig* 17 • Immunoelectrophoretic patterns of A.ceylanicum E-S

products collected at 2 hr ( a ) , 4 h ( b ) , 6 h (c) and

8 h (d) using rabbi t anti- / \cAserum.

82

0.20 O.AO 0.60 0.80

Antigen concentration ( / jg /we l l )

1

1.0

Fig. 18: Effect of different concentrations of excretory-secretory products from A.ceylanicam adults on the specific activity in enzyme linked immunosorbent assay using 1:500 (X-X); 1:1000 (0-0) and 1:2000 (A-A ) dilutions of rabbit anti-E-S serum.

83

E-S antigen, the antibody t i te rs of parasite specific antibodies

in all the three immune rabbit sera (rabbi t anti-E-S, rabbit

anti-Ac A and rabbit anti-AcL) were determined and the t i t r a ­

tion curves are shown in Fig. 19. All the three titration

curves of E-S products obtained with rabbi t anti-E-S and

rabbit anti-adult and rabbit anti-larval sera were parallel

and showed linearity upto a serum dilution of 1/16000. A

reciprocal ELISA t i te r value of 256,000 was obtained for rabbit

anti-E-S, while rabbit anti-AcA and rabbit anti-AcL sera showed

an antibody t i te r of 128,000.

ANTIGENIC ANALYSIS OF E-S PRODUCTS

The E-S products preparation in Ringer's solution

were analysed by immunoelectrophoresis (lEP) using rabbit

anti-E-S serum which showed the presence of 8 precipitin

bands in E-S products (Fig. 20). The E-S products prepared

in other two media (RPMI-1640 and DMEM) were alao analysed

in lEP with rabbi t anti-E-S serum and almost the same number

of precipit in bands (7-8) were observed with slight differences

in the intensities of certain precipitin bands (Fig. 20).

The E-S products were further analysed by crossed

immunoelectrophoresis (CIE). The CIE pattern of E-S products

using rabbi t anti-E-S serum is shown in Fig. 21a and the

diagrammatic representation of the same is given in Fig.21b.

Strong antigenic peaks are shown by solid lines whereas the

broken lines indicate that the faint antigenic peaks. Each

84

8 o

o o

o

o - o o

c o 2 •5

E

o

"ifl u O w

a ij cr

1 ^

S -P " 0

rj -H C

'2 4' = £ ^ E

/ - . 4) +jT3

? 1 ^ ^ « 5

0.^ W5 (U

W O >.

c n! <iJ

n) C > if>

•*^ •*-• ^

(Z6VaO) '^H'^IPP DijrDads •H

85-

Fig. 20" Immunoelectrophoretic patterns of A.ceylanicum E-S

products prepared in three different media; Ringer's

solution ( a ) , RPMI-1640 (b) and DMEM (c) , using rabbit

anti-E-S serum.

2€

1

•

'

• -

V

J ^ y

- ^ —^

y

Fig. 21: Crossed immunoelectrophoretic pattern of A.ceylanicum

adult E-S products using 10% rabbit anti-E-S serum

(a) Protein stained pattern, (b) Diagrammatic represent­

ation. Solid lines represent strong antigenic peaks.

Arrows indicate the point of application of antigen.

Anode ( + ) is to the right in first dimension and to

the top in second dimension.

87

antigen is denoted by a single peak and CIE revealed 16

antigens in A. ceylanicum E-S products, 13 moving towards

anode and 3 towards cathode.

Common antigens between the E-S products and the somatic

extracts

In order to identify the common antigens between

the E-S products and the somatic extract from adult and larval

stages of A. ceylanicum, the E-S products were analysed in

lEP with sera raised against the somatic antigens. Six and

4 precipitin bands were observed in E-S products with rabbit

anti-AcA and rabbit anti-AcL serum respectively (Fig. 22),

Almost the same number of antigens were detected in somatic

extracts from adult and larvae, when tested in lEP against

the rabbit anti-E-S serum (data not shown). The E-S products

were further analysed by CIE with sera raised against somatic

antigens. CIE analysis of E-S products with rabbit anti-adult

serum showed the presence of 12 antigens, out of which 11

antigens were anodic in nature and one antigen moved towards

the cathode (Fig. 23). When rabbi t anti- larval serum was

employed in CIE and 6-7 antigenic neaks were

observed in E-S products and all of them appeared to be

anodic (Fig. 24). The results of lEP and CIE analysis of

E-S products with immune rabbit sera are summarised in Table

11. The common antigenic determinants between the E-S pro­

ducts and somatic extracts from adult and larval stages of

8S

Fig. 22: Immunoelectrophoretic patterns of A.ceylanicum adult

E-S products with rabbit anti-E-S serum (1) , rabbit

anti-adult serum (2) and rabbit anti-larval serum (3) .

89

Fig. 23: Crossed immunoelectrophoretic pattern of A.ceylanicum

adult E-S products using 10% rabbit anti-adult serum

(a) Protein stained pattern, (b) Diagrammatic represent­

ation. Solid lines represent the strong antigenic peaks

Arrows indicate the point of application of antigen.

Anode ( + ) is to the right in the first dimension and

to the top in second dimension.

90

Fig. 24: Crossed immunoelectrophoretic pattern of A.ceylanicum

adult E-S products using 10% rabbit anti-larval serum

(a) Protein stained pattern, (b) Diagrammatic represent­

ation. Solid lines represent the strong antigenic peaks.

Arrows indicate the point of application of antigen.

Anode ( + ) is to the right in first dimension and to

the top in second dimension.

91

3

nl

B s

<H U a

i 4) U

•»-> u 3

0

a I

to

1—I

c

c • H

c <

ni 0)

a tn

tJ c rt

X!

•H

0 o

0

0)

B 3 z

E 3

in •rt c <

B «i OC

• H

c <c

••

V I - t jQ ,« H

<u 3

• H

c ,n o 0)

H

u •H

C

••J

m O

CO I—1

o •H

c

t/3 I

w

(0 •H tn

u o

X!

0 o c u 3 -*->

1 <" I—( 0)

l-H t ^

E 3 u (U to

Ul 1 1 1

• H + J c A

• H

^ 2 rt oe;

e 3 v< 0) to

< • < j

.A • p

c «)

• H J3 X) n)

oe;

E 3 U V to

J u < 1 • H

4->

c rt'

• H J3 JD (d

OS

E 3 >H 0) to

Vi 1

W 1 •1-1 4 J

c ni

•H ja Xi Rt

CHS

E 3 U 4) to

< U

< 1 • H

+ J c nl

•H Xi XI (6 cc

E 3 U 1) (0

J o < 1 1

•H + J

c n)

•H J3 ^ «)

OS

I w

0 c 3 H E

•H

0) to to O U

U

to • H to (1)

0 X! a o u +> u 9) t)

I I r

•»->

u .•« )4

*> X 4)

u •1-1 ••J

«) E 0 to

1—1 3

T3 R)

B 9 U

1 •< V u •

< II

< V <

••J u nt U

•»->

X 4)

U • H ••J

(« E 0 to

r—1

n) > nl

r - t

B a u • H

J •^ 0) u •

1 < II

J o <

3

e o >H

«*H

to •«-> u 3

T3 o u a.

>^

0 •*->

4) o 4J to 1 > u 0

4^ t) u V X

1 w II

t/2 1

w

92

A.ceylanicum were further analysed by doing Tandem crossed

immunoelectrophoresis (TIE) of E-S products with adult (AcA)

and larval (AcL) somatic extracts and the TIE patterns are

shown in Fig. 25. On comparing the TIE pattern of E-S pro­

ducts plus AcA with CIE pattern of E-S antigens (Fig.2^0)

11 antigens were found to be common with adult stage (Fig.

25 b ) . TIE analysis of E-S products plus AcL is shown in

Fig. 25a, which revealed the presence of 6 common antigens

between the E-S products and the larval somatic ext rac ts .

Presence of host serum proteins in E-S products

In order to detect the presence of host proteins in

E-S products hyperimmune rabbit serum was raised against

normal hamster serum (NHS). When E-S products were reacted

with rabbi t anti-NHS serum in CIE, 6 antigenic peaks were

observed (Fig. 26), suggesting thereby the presence of host

proteins in the excretory-secretory products of A.ceylanicum

adults .

IMMUNISATION OF HAMSTERS WITH EXCRETORY-SECRETORY PRO­

DUCTS AND EVALUATION OF IMMUNISED HAMSTERS FOR PRO­

TECTIVE IMMUNITY

In view of potential importance of E-S products of

parasit ic nematodes including hookworm parasi tes in develop­

ment of protective immunity, hamsters were immunised with

E-S antigens from adult A.ceylanicum followed by subsequent

95

< . ^

t

V

^

E T3 c 0)

c n)

u CD

+-> o 3

t! O

a V2

I

w

3

nt

B s u

u

c u 0)

m

a

^ o x; a o u + j

o 0)

.—( (U o c 3 E E

T3 (U

(n O

O

IT)

no c nt

1i ^ c o o ^

J3

u

•t->

X 0)

3

u 3

T ) O J-.

a I

w <4-4

o

c u t)

+->

a

nt o

C

CO

o

<

O

E 3 u V 10

C/1 I

w I

c

nj +

C T3

to c => <

0)

o X! a o u +-> o v

I — I

(U o c 3 E E

<D (0 U] o u u

0)

E o m

>

c «)

c o

•H to c i) E

CO

x: bC

4) X

C 0)

in ac u

c

c o

s a

c o

•H CO c 1) E

•H Tl 73 c o o i) CO

O -H

• H

4-

i ' ^

Fig. 26: Crossed immunoelectrophoretic pattern of A.ceylanicum

E-S products using rabbit anti-normal hamster serum.

Arrows indicate the point of application of antigen.

Anode ( + ) is to the right in the first dimension and

to the top in second dimension.

95

challenge infection. The immunised hamsters were bled 15

days after the first injection. Further immunisation and

bleeding was done alternately at weekly intervals and sera

obtained after each injection were tested by enzyme linked

immunosorbent assay (ELISA) for the presence of parasite

specific antibodies. The antibody t i t e r s of hamster anti-

E-S sera (obtained after every injection) are expressed as

geometrical mean reciprocal t i te r (GMRT) and are given In

Table 12. No antibody could be detected in the serum obtained

after 1st injection. A GMRT value of 50.00 was obtained

after the 2nd injection which increased to GMRT values of

200.00 and 303.14 after the 4th and 5th injections respectively.

The presence of precipitin antibodies in immune hamster sera

was analysed by immunodiffusion (ID). No precipitin bands

could be observed in sera taken after first injection while

one and 2 precipitin bands were observed in ID after the

2nd and the 3rd injections respect ively . The number of preci­

pitin bands increased to 3 after the 4th injection and remained

constant after that ( i . e . after 5th injection). The results

of ID analysis are summarised in Table 13. The immune hamster

serum obtained after the 5th injection was theln tested in

lEP and 4 precipit in bands were observed in E-S products

(Fig. 2 7). The immune hamsters were then evaluated for

protective immunity by challenging them with an oral dose

of 60 L- one week after the fifth injection. The percent

reduction in worm burden at 21 days post challenge showed

96

nl (0 (fl n)

c V

O (n o c 3 E E

T3 (U

c

E N

c

>

u

(n E nl

J5

9) c 3 E E

0)

O JD •H +J B

<

(M

n) H

c o

4J ft)

c o

•H

u J3

>

H OS S o

0)

u

I <

T3 ^

C

o •H

o 0)

c

B

o

•*

o

o o

• o o (M

i n

c c •H

c

E 3 0) (0

• H -t-> c <

B 0) M

•H

B

<

O o o i n

Q Z

I

I

B <

<U • » - '

cn E m X

4) >

E o u

T3 0) B

J3 O

0)

I W

(0 •(->

u 3 73 0 h 0.

>-u o

•t-> 0) >H

o 0) cn 1 > -u o ••-> 0)

o X W

II

V5 1

W

nt u 0 U

a •H

u 4) U

B

0)

E i-H n) u

•H

0) E 0

(0 «)

73 V (0 (0 4J

a X

• (0 B 0}

• H + J B n)

C/2 1

U

0) JC ^ nl

(0

- t j • H

^ 4) TJ ^

•!-> •H •t-»

0) N

• H B 3

>- E 73 0

J3 • H

-(-> B n)

0) X

E • H

(0 ^4

m E n)

H Xi

• V

r—1 JD n)

-t->

u 0)

+ J 4}

13

+ j

o B

^ TJ 0

J3 •H + J B < II

Q 2

97

•g

3 T3

E o u

u

3

0

a C/2

I

• H (fl t>-

—) nt c n)

c o

E 3 U 0) en

t/2 1

W

3

O C 3

e B

c

u 0)

-t-" tn E n)

X !

en

c

o d)

a O

0) JD

E 3

z

E 3 U V (A

•rt c <

c

• H 4-> C

<

c o

> 0>

c o

+» u > 0)

c

I—I i)

T( B

1—( t-H

•!->

in t-H

•;3 o 4)

c" •H

c 0

•H 4-* o 0)

E 3 U i) (0

{/3 I

w I

•H •(-> c rt

0)

(fl E M

I

w

(0 V< 4)

•t-> (0

E n)

u n)

u n) 0)

B O

U 0)

43 o nt

(U

0)

c o

1)

(A

0) ••-» (Q

E n)

J3

c 0)

E •H

K X 0)

0) 0)

u

c 4)

E

x: (U Vi cd

D

>

(0 4) 3

.—I nt >

(U N

•H B 3 E E

B a u

•H

I H V u <l 3

T) nt

E o u

u 3

T ) O

> •

U

o (U u u 0) (A I >~ u o

u u X w

II

(U

92>

Fig. 27: Immunoelectrophoretic pattern of E-S products from

A.ceylanicum adults using hamster anti-E-S serum.

100

the degree of protection. Seventy five percent reduction

in worm burden could be observed as compared to the un-

immunised infected controls (Table 14).

DISCUSSION

The release of antigenic molecules (excretory-secretory

products) during jii^ vitro incubation has been well documented

for a wide variety of nematode parasi tes v i z . , T. spiral is

(Despommier and Muller, 1976; Parkhouse and Clark, 1983;

Silberstein and Despommier, 1985; Gamble, 1985; Capo et a l . ,

1986), T.canis and^ T.cati (Sugane and Oshima, 1983; Maizels

et al^., 1984; Badley 1987a,b; Kennedy et al^., 1987a), T.muris

(Jenkins and Wakelin, 1983), A.suum and A.lumbricoides (Urban

and Romanowski, 1985; Kennedy and Qureshi, 1986; Kennedy

e ^ a l . , 1987b), N.americanus (Carr and Pr i tchard , 1986, 1987)

and H. poly gyrus (Ey 1988a,b).

The JL£ vi t ro released or the excretory-secretory

(E-S) products of parasit ic nematodes including hookworms

have been shown to be more immunogenic as compared to soma­

tic antigens (Thorson, 1951; Campbell, 1956; Chipman, 1957;

Katiyar et al^., 1972; Govila et al^., 1973; Poulain et al^.,

1976; Murray et al^., 1979; Silberstein and Despommier, 1984;

Mimori et al_., 1987). In spite of their less complex nature,

relatively l i t t le effort has been made to evaluate critically

101

the potential usefulness of E-S antigens in inducing protective

imnnunity to hookworm parasi tes and also to analyse the anti­

gens present in these products.

In the present study, we have optimised the conditions

tor preparing the E-S products from adult hookworm parasite

A.ceylanicum and have analysed the E-S products using hyper­

immune rabbit sera employing certain immunochemical techniques.

The conditions for the short term iji vitro maintenance of

adult A.ceylanicum and preparation of E-S products were stand­

ardised in order to get the maximum viabil i ty of the worms

during in vitro incubation and also the maximum yield of

E-S products. The time for ^ vi t ro incubation was also

optimised and was- found to be 6 h . The motility of the

worms started decreasing when the worms were kept for the

longer period. The parasite could not be maintained beyond

8 h even after transferring them into the fresh medium. A

period of 6 h for jji vi tro incubation of worms was chosen

in order to avoid the contamination of E-S products with

the products from the dead and disintegrating parasi tes .

Since there was not any significant difference in the amount

of protein released as well as the antigenic pattern of E-S

products prepared in the three media (Ringer's solution,

RPMI-1640, DMEM), Ringer's solution has been chosen to prepare

the E-S products because this is much simpler (containing

only salts and glucose) and more economical medium as compared

to RPMI-1640 and DMEM. The amount of protein released

102

by A.ceylanicum adults in 6 h (125±25 ug/100 adults) was

in accordance with other findings by Rotmans et ajl_. (1981),

who have also shown the release of 100 ug E-S products per

100 worms of Schistosoma mansoni by maintaining them in

M 199. Minute quantities of E-S products have been shown

to be released by the larvae of T.canis (0.8 ug/100 larvae/day)

and H.polygyrus ( 20 ng/100 larvae/day) during in vitro incu­

bation (Badley et_ a l . , 1987a; Ey, 1988). The human filarial

parasi te: Brugia malayi has been shown to release 18 ug of

E-S products/100 adult worms (Kaushal et al_., 1982). However,

considerably high amount of E-S products (12-15 mg/100 adult

worms) were released by the bovine filarial parasite Setaria

cervi (Malhotra et al_., 1987).

The t i t e r s of parasite specific antibodies in rabbit

anti-E-S serum were quantitated by ELISA. This test has

been used ear l ier for detecting the antibodies against nematode

parasites including hookworms (Wedrychowicz et al^., 1984;

Roach e^al^. ,1988; East et_ al., 1988). The rabbit sera raised

against E-S products gave quite a high antibody t i ter and

the t i ter with anti-E-S serum was one dilution higher when

compared to anti-adult and anti- larval sera. The highti tered

hyperimmune rabbi t anti-E-S serum was used to analyse the

antigens present in E-S products of A. ceylanicum adults by

immunoelectrophoretic techniques. The antigenic analysis by

lEP revealed the presence of 8 precipitin bands in A.ceylanicum

E-S products. Almost same number of antigens (7 precipitin

103

bands) have been reported in the E-S products of S.nyansoni

(Rotmans et al^., 1981), A.cantonensis (Techasoponmani and

Sirisinha, 1980) and S.cervi (Malhotra et al^., 1987) using

lEP. NKU et al^. (1981) could detect at least 4 precipitin

lines in E-S products of 0 . volvulus mf with patient serum

in lEP. Antigenic characterization by CIE resulted in better

resolution of A. ceylanicum E-S products. Out of different

serum concentrations t r i ed , 10% concentration of immune rabbit

serum was found optimum for the better separation of antigenic

peaks by CIE. On decreasing the antiserum concentration,

the faint antigenic peaks disappeared while the use of higher

serum concentration resulted in the poor resolution of certain

antigenic peaks . A total of 16 antigens were identified in

the E-S products of adult A.ceylanicum out of which 8 were

found to be major. The E-S products of A.ceylanicum appeared

to be less complex in comparison to the somatic antigenic

preparation from adult stage as it revealed the presence of

34 antigens in CIE. The E-S products of A.ceylanicum may

contain some heterogenous antigens as indicated by the doubling

of antigenic peaks 11, 12. This could be due to a number

of reasons viz; the presence of isoenzymes genetic variants

of the same protein, or protein complexes and their monomers

or the presence of molecular heterogeneity deriving from

different degree of glycosylation.

The E-S products appeared to share antigenic determi­

nants with the somatic antigens from adult and infective Jarval

104

stages of A.ceylanicum as shown by the reactivity of E-S

products with rabbit sera raised against the somatic antigens.

Twelve (nos.2-5, 7-13, 16) and 7 (nos. 1-3, 5, 8-10) antigens

from a total of 16 antigens of E-S products were recogined

bv the rabbit anti-adult and rabbit anti-larval serum respect­

ively. The presence of common antigenic determinants between

the E-S products and somatic extracts from adult and larvae

of A.ceylanicum was further confirmed by doing Tandem crossed

immunoelectrophoresis, which also showed almost the same

number of common antigens. Antigen sharing between E-S pro­

ducts and the somatic antigens has been observed for a number

of paras i tes . Kaushal et_ al_. (1982) have reported that most

of the E-S antigens_ of B.malayi adults were common to those

present in the somatic extracts of adult worms. Antigenic

sharing has also been reported between the somatic extracts

and E-S prodoucts of L.carinii (Ishii 1980) , F_. hepatica

(Klimenko, 1977), A.cantonensis (Techasoponmani and Sirismha,

J980), S.vulgaris (Wynn et_ a^., 198i) and Trichuris muris

(Jenkins and Wakelin, 1977; 1983). Commonness of antigens

between the somatic extracts from adult and microfilariae

and E-S products of female adult S.cervi has also been ^hown

employing immunoelectrophoretic technique (Malhotra e£ al_.,

1987).

Parasitic worms are known to absorb the host oroteins

on their surface to evade the immunologically hostile environ­

ment of the host (Smithers et a l . , 1969). The adults of

105

A. ceylanicum worms reside in the submucosal layer of small

intestine and are voracious blood suckers, there is a high

probabili ty of absorption of serum proteins bv the worms

and these proteins would subsequently be released during

in vitro maintenance ot the worms. Hyperimmune rabbit serum

against normal hamster serum (NHS) were prepared to detect

the presence of host serum proteins in E-S products.

CIE of A. ceylanicum E-S products with rabbit anti-normal

hamster serum showed the presence of 6 precipitin peaks

thereby indicating the presence of host proteins in E-S products.

The presence of host proteins in the E-S products of different

parasites have been reported by several workers (Kaushal

et^al^., 1982; Maizels et_ a]_., 1984; Harnett et a^., 1986: Malhotra

et a l . , 1987).

There is substantial evidence indicating the protective

potentials of E-S antigens of some nematode parasites including

hookworms (Thorson, 1951; Katiyar et_ al^., 1972; Govila et_

al^., 1973; Poulain et_ a]_., 1976; Sugane and Oshima, 1983;

Jenkins and Wakelin, 1983; Maizels et_ al^., 1984; Badley e^

al_., 1987a,b; Ey, 1988). In the present study, the E-S anti­

gens of A. ceylanicum adults have been found to stimulate a

significant level of protective immunity as the hamsters immu­

nised with E-S products showed 75% reduction in worm burden.

Wedrychowicz et_ al^. (1984) have reported 69% protection bv

E-S products from adult N.brasi l iensis . The antigens obtained

from cultures of A.duodenale (Jain et_ al^., 1979), T.earns (Sugane

106

and Oshima, 1983; MaizeLs e; al^., 1984; Badley et al^-. 1987a,b)

and T.muris (Jenkins and Wakelin, 1983) have also been shown

to induce part ial resistence against infection with the respective

paras i tes .

In conclusion, the excretory-secretory (E-S) products

from A.ceylanicum adults could be obtained by short term

in vitro maintenance of adult worms in Ringer's solution which

is much simpler and economical medium. The rabbit anti-

E-S sprurri having high t i te rs ot parasite specific antibodies,

showed the presence of 16 antigens in the E-S products. Twelve

and 7 antigens of E-S products shared antigenic determinants

with adult and larval somatic extracts respect ively. Some

of the antigens of ' E-S products are derived from the host

serum proteins. The protective potential of E-S products

has also been evaluated and the hamsters immunised with

E-S products showed significant level of protection. A logical

extension of these studies would be to characterise the E-S

products on molecular basis in order to identify the protective

antigens.

CHAPTER lY

SUMMARY AND CONCLUSION

107

Ancylostomiasis is one of the major public health problems

in tropical countries. The world incidence of hookworm infect­

ions are 1000 million with the death rate of 55 thousand per

year. The currently available anthelmintics are not very

effective. This clearly emphasises the need to intensify

the control measures in hookworm endemic areas . An alternate

approach for the permanent control of the disease would be

to develop immunoprophylactic measures against hookworm

paras i tes . However, the existing knowledge about the molecular

immunology of hookworm infection is s t i l l in i ts infancy.

Hookworm parasi tes evoke functional protective immunity and

some reports are available regarding the protective potential

of live parasit ic stages as well as of somatic extracts and

excretory-secretory products from adult and larval stages,

however, not much is known about the molecules/antigens which

are involved in evoking these protective responses. A basic

knowledge about the total antigenic composition of the para­

sites is essential not only for understanding the host parasite

relationships but also for the identification of antigenic ta r ­

gets of protective immunity. We have, therefore, directed

our studies towards the characterisation of antigens from adult

and larval stages of human hookworm parasi te ; Ancylostoma

ceylanicum, which has earl ier been used for the screening

ot anthelmintic compounds by several workers. TotaJ proteir)

and antigenic composition of adult and larval stages of

A. ceylanicum were studied employing SDS-polyacrylamide gel

108

electrophoresis (SDS-PAGE) and immunoelectrophoretic techniques

using hyperimmune rabbit sera . The levels of parasite specific

antibodies in immune sera were determined by enzyme linked

immunosorbent assay (ELISA). Similar techniques were used

for antigenic analysis of the excretory-secretory products

from adult A.ceylanicum. The protective potential of somatic

extracts from adult and larvae as wellas the excretory-secretory

products from adult A.ceylanicum was also studied by immunising

the naive hamsters with these antigens (somatic extracts from

adult and larvae and excretory-secretory products from adults)

toilowed b \ challenge infection.

The somatic extracts from adult (AcA) and larval stages

(AcL) of A.ceylanicum were separated on SDS-polyacrylamide

gradient gel electrophoresis (SDS-PAGE), which showed the

presence of 47 protein bands in adults and 43 in larvae in

the molecular weight range of 10-170 kD. Most of the protein

bands were found to be common between these two stages

(adult and la rvae) , however, 8 (130, 125, 42, 30, 20, 17,

16.5 and 10 kD) and 4 (170, 160, 153 and 85 kD) protein

bands specific to adult and larval stages respectively were

observed. The quantitation of parasite specific antibodies

in immune rabbit sera was done by enzyme linked immunosorbent

assay and reciprocal antibody t i t e r s of 256,000 and 128, 000

were obtained for rabbit anti-adult and rabbit anti-larval

sera respectively using AcA antigen at optimum concentration

of 0.25 ug/well. Both these sera showed the reciprocal anti-

109

body t i t e r s of 256,000 with AcL at optimum concentration

of 0.50 ug/well.

The antigenic analysis of somatic extracts from adult

and larval stages of A.ceylanicum was initially done by Immuno­

electrophoresis ( lEP), which revealed 10-12 precipitin lines

in AcA with rabbi t anti-AcA serum and 6-7 precipitin bands

in AcL with rabbit anti-AcL serum. Further characterisation

of these somatic antigens was carried out employing crossed

Immunoelectrophoresis (CIE) which showed the presence of

34 antigenic components in AcA and 19 antigens in AcL. Out

of 34 AcA antigens, 25 were anodic and 9 cathodic while 17

anodic and 2 cathodic antigens were observed in AcL. In order

to study the cross-reactive or common as well as stage/parasite

specific antigens of A.ceylanicum, crossed Immunoelectrophoresis

employing heterologous sera and crossed line immunoelectro-

phoresis (CLIE) were used. When AcA and AcL were reacted

with heterologous immune rabbit sera in CIE, most of the

antigens were found common or cross-reactive between the

two stages, however, 12 antigens specific to adult and 5 anti­

gens specific to larval stage were observed. The subsequent

analysis of CLIE also revealed the same number of common

or cross-reactive as well as stage specific antigens in AcA

and AcL. In order to analyse A.ceylanicum antigens having

cross-reactivi ty with rat hookworm - Nippostrongylus brasi l iensis ,

CIE using immune rabbit sera raised against adult (NbA) and

infective larval (NbL) somatic extracts of N.brasiliensis and

110

CLIE using NbA and NbL in the in te rmedia te ge l , were done.

Most of the antigens (22-26) were found to be AcA spec i f i c ,

only 12 and 8 antigens of AcA were found to be common/cross

r e a c t i v e wi th NbA and NbL r e s p e c t i v e l y . On analysing the

c r o s s - r e a c t i v e or common antigens of AcL with NbL and NbA,

13 common or c ross r e a c t i v e and 6 AcL specif ic antigens were

o b s e r v e d .

Another set of antigens analysed in the p resen t study

i s t he e x c r e t o r y - s e c r e t o r y (E-S) p roduc t s from adult

A. cey lanicum. The condit ions for t h e p repa ra t ion of E-S

p roduc t s were opt imised with r e s p e c t to medium as well as

the incubation t i m e . Out of t h r e e different media; RPMI-1640,

DMEM and R i n g e r ' s solution t r i e d . R inge r ' s solution proved

to be the bes t as the mot i l i ty of t h e worms was maximum

in t h i s medium and t h e worms could be maintained upto 6h

without changing t h e medium. The amount of prote in re leased

was found to be 95±15 ug/100 adults in RPMI-1640 and DMEM

as compared to 125±25 ug/100 adult worms in R inge r ' s solut ion.

Immunoelectrophoret ic ana lys i s using r a b b i t anti-AcA serum

showed the presence of 5-7 p r e c i p i t i n bands in E-S products

p r epa red in t h e s e media . As t h e r e was not any significant

difference in t h e amount of pro te in r e l ea sed as well as the

antigenic p a t t e r n s of E-S p roduc t s p r e p a r e d in t h r e e media .

R inge r ' s solution (containing s a l t s and glucose) was chosen

to obtain t h e E-S p roduc t s from A»cey lanicum adu l t s because

t h i s i s much s imple r and more economical in comparison to

o the r two media (RPMI-1640 and DMEM).

I l l

A polyvalent hyperimmune rabbit serum was raised dgainst

A.ceylanicum adult E-S products and the t i ter of parasite

specific antibodies in immune rabbi t sera was determined

by enzyme linked immunosorbent assay using optimum concentrat­

ion of 0.50 ug E-S antigen/well. A reciprocal antibody ti ter

of 256,000 vras obtained with rabbit anti-E-S serum. The

E-S antigens were also tested with the immune rabbit sera

raised against somatic extracts of A.ceylanicum in ELISA and

a t i ter value of 128,000 was obtained for both rabbit anti-

adult and rabbit anti-larval sera . Crossed Immunoelectropho­

resis (CIE) using rabbit anti-E-S serum revealed 16 antigens

m A.ceylanicum E-S products, out of which 13 were anodic

and 3 moved towards the cathode. E-S products have also

been found to share antigenic determinants with the somatic

extracts from adult and larval stages. lEP analysis of E-S

products showed the presence of 6 and 4 precipitin bands

with rabbit anti-AcA and rabbit anti-AcL sera respectively.

On further analysing the E-S products by CIE using rabbit

sera raised against somatic extracts 12 and 7 antigens of

E-S products were found to share antigenic determinants with

AcA and AcL respect ively. Tandem crossed Immunoelectropho­

resis (TCIE) of E-S products with AcA and AcL also revealed

almost the same number of common/cross-reactive antigens

between the E-S products and the somatic ext rac ts . The pre­

sence of host serum proteins in the E-S products was also

analysed by CIE using rabbit serum raised against normaJ

112

A polyvalent hyperimmune rabbit serum was raised against

A.ceylanicum adult E-S products and the t i ter of parasite

specific antibodies in immune rabbi t sera was determined

by enzyme linked immunosorbent assay using optimum concentrat­

ion of 0.50 ug E-S antigen/well. A reciprocal antibody t i ter

of 256,000 v/as obtained with rabbit anti-E-S serum. The

E-S antigens were also tested with the immune rabbit sera

raised against somatic extracts of A.ceylanicum in ELISA and

a t i te r value of 128,000 was obtained for both rabbit anti-

adult and rabbit anti- larval sera . Crossed Immunoelectropho­

res is (CIE) using rabbit anti-E-S serum revealed 16 antigens

in A.ceylanicum E-S products, out of which 13 were anodic

and 3 moved towards the cathode. E-S products have also

been found to share antigenic determinants with the somatic

extracts from adult and larval stages. lEP analysis of E-S

products showed the presence of 6 and 4 precipitin bands

with rabbi t anti-AcA and rabbit anti-AcL sera respectively.

On further analysing the E-S products by CIE using rabbit

sera raised against somatic extracts 12 and 7 antigens of

E-S products were found to share antigenic determinants with

AcA and AcL respect ively. Tandem crossed Immunoelectropho­

resis (TCIE) of E-S products with AcA and AcL also revealed

almost the same number of common/cross-reactive antigens

between the E-S products and the somatic ext rac ts . The pre­

sence of host serum proteins in the E-S products was also

analysed by CIE using rabbit serum raised against normal

113

hamster serum, and 6 antigens in the E-S products appeared

to be of host origin.

The protective potential of somatic extracts from adult

and larval stages as well as excretory-secretory products

trom ddult A.ceylanlcum was tested by immunizing the hamsters

with these antigens and evaluating the immunized hamsters

for protective immunity. The sera obtained from immunized

hamsters, before challenge, showed significant levels of para­

site specific antibodies. The immunodiffusion and immuno-

electrophoretic analysis revealed the presence of precipitin

antibodies in immune hamster sera and 7 precipitin bands

in adult somatic extracts and 4 in both larval somatic extracts

and E-S products. -The hamsters immunized with larval somatic

extracts showed maximum protection (93% reduction in worm

burden) while 75% and 63% protection was observed in hamsters

immunized with excretory-secretory products and adult somatic

extracts respect ively.

Therefore, in the present study we have been able to

characterise the protein and antigenic components of adult

and larval stages of A.ceylanicum using hyperimmune rabbit

sera having high t i t e r s of parasite specific antibodies and

certain Diochemicai and Immunochemical techniques. The common

or cross-reactive antigens between the two life stages of

A.ceylanicum and also between the two hookworm parasites

(A.ceylanicum and N.brasiliensis) as well as stage and parasite

114

specific antigens have also been identified. We have been

able to prepare the excretory-secretory products of A.ceylanicum

adults using Ringer's solution which is much simpler and

a more economical medium. Antigenic characterisation of E-S

products revealed less complex nature of these products as

compared to the adult somatic ext rac ts . The protective poten­

t ials of the somatic extracts from adult and larval stages

and also of E-S products from adult A. ceylanicum was also

studied. The larval somatic ext rac ts , though contain less

number of antigens, showed maximum protection followed by

excretory-secretory products and adult somatic extracts .

These studies provide basic information about the antigenic

compositions of somatic extracts and excretory-secretory pro­

ducts of A. ceylanicum as well as the comparative efficacy

of these antigens in inducing protective immunity against this

paras i te . However, further studies on molecular characterisa­

tion of these antigens are certainly required for identifying

the antigenic targets of protective immunity.

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