STUDIES ON CHANDLERELL HAWKiNCfA , A FILARIA PARASITL …ir.amu.ac.in/1891/1/T 5322.pdf · ABSTRACT...

STUDIES ON CHANDLERELLA HAWKiNCf, A FILARIAL PARASITE OF CROW

ABSTRACT

Mthesis SUBMITtED' '

IN FULM.LMENT:OF THE:RHQUIREMENTS FOR THE DEGREE OF

DOCTOR .OF PHlLOSdPHY

I N .

/ n o m R Y

SHAHNAZ BANO

SECTION OF PARASITOLOGY DEPARTMENT OF ZOOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH

January, 1981

ABSTRACT

The thes i s embodies the r e s u l t s of the s tud i es

on Chand l e r e l l a hauking i , a f i l a r i a l p a r a s i t e of Ind ian

jung l e crou, Corvus macrorhynchos ( U a g l e r ) . Only f our

a s p e c t s , morphology, h i s t o l o g y , h i s t ochemis t r y and

i n ^ i t r o cu l ture have been taken up.

The morpho log i ca l s tud i es inc lude a re d e s c r i p t i o n

of adul t worm u i th an add i t i on of a feu minor d e t a i l s

in the adu l t . The s t ruc tu r e s descr ibed in m i c r o f i l a r i a

are the nuclear s t r u c t u r e s , c epha l i c s t r u c t u r e s ,

pharyngea l thread , Innenkorper and a l so occurrence of

tuo forms of m i c r o f i l a r i a e in blood of crou.

The nuclear s t ruc tu res naue been s tud ied u i th

s p e c i a l r e f e r ence to nuclear landmarks which are of

g r ea t taxonomic vyalu^.

Sometimes the nuclear s t ruc tu r e s do not s u f f i c e

f o r i d e n t i f i c a t i o n of genera and s o e c i e s , the c e p h a l i c

s t r u c t u r e s , pharyngeal thread and Innenkorper have a l so

been s tud i ed .

I t has a lso been proved that the tuo forms of

m i c r o f i l a r i a e , the long and the s h o r t , present in the

blood of crou belono to the same s p e c i e s , C. hauking i .

The s te reoscan s tud ies of the c u t i c u l a r s t ruc tu r e s

of the adult and the m i c r o f i l a r i a have heen done as these

are of g rea t taxonomic importance in c l a s s i f i c a t i o n of

uorms.

The h i s t o l o g i c a l s tud i es inc lude the study of

h i s t o l o g i c a l f e a t u r e s of the body u a l l , musculature,

a l imentary canal and reproduct iue organs as some of these

s t r u c t u r e s prov ide a taxonomic t o o l at var ious l e v e l s .

Among h i s tochemica l •^^tudies, four enzymes, v i z ;

ac id phosphatase, a l k a l i n e phosphatase, adenosine

t r i phospha tase and c a r b o x y l e s t e r a s e have been l o c a l i z e d

as these enzymes are a t t r i b u t e d to d i f f e r e n t func t i ons

and t h e i r d i s t r i b u t i o n i s very s p e c i f i c . Thus the

l o c a l i z a t i o n of these enzymes helos in determining the

va r i ous func t i ons ass igned to d i f f e r e n t organs and par ts

of the body of adult uorm.

In m i c r o f i l a r i a , f i v e en zy r e s , ac id phosphatase,

a l k a l i n e phosphatase, adenosine t r i phospha tase ,

carboxy l e s t e r a s e and a r y l su lphatase , have betin s t u d i e d .

The d i s t r i b u t i o n of these enzymes i s so s p e c i f i c that

t h e i r pa t t e rns help in i d e n t i f i c a t i o n nf d i f f e r e n t

genera and s p e c i e s .

In the present uork only m i c r o f i l a r i a e of

C. haukingi have been CLi l t ivated in v i t r o , in v i eu to

study t h e i r development outsidf-i the body of the hos t .

These s tud ies are important even f o r t e s t i n g the drugs

on p a r a s i t e a lone and a l so f o r c o l l e c t i o n of ES products

uhich may servs as f u n c t i o n a l ant igens aga inst

f i l a r i a s i s .

STUDIES ON CHANDLERELLA HAWKING!, A FILARIAL PARASITE OF CROW

. A THESIS SUBMITTED;^ IN FULFILMENT OF THE REQtjiREMENTS

FpR THE DEGREE OF . .

DOCTOR OF PHILOSbyHY IN

ZOOLOiSY

BY

SHAHNAZ BANO

SECTION OF PARAiil ' l 'OLOUY

DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY

ALIGARH

January, 1981

/ J

T5322

l l i p r i ) M u s l i m ALIGARH. M. P. 202001

^Enibersiitp PARAS ITOLOGY RESEARCH L A B O R A T O R Y

DEPARTMENT OF Z O O L O G Y

Director ; Ather H. Siddiqi Ph.D. (Aiig.); Ph.D. (Purdue) 5 3anuary, 1981

This i s to cert i fy that the thesis entitled, "Studies on Chandlerella hamkingi« a f i l a r i a l parasite of crow", which is beirrg submitted by Tslrs. Shahniaz Banoi, embadies original urork done by the candidate herse l f . The entire toark was carried out under my stipervisipn. betuieenB 1976-1980 and that I alloui her ta submit the same in fulfi lment of the requirements for the degree of Doctor of Philcisophy in Zoology of this University.

Q \

ATHER W. SIDDIQI Supervisor

ACKNOULEDGEI ENTS

I uish to extend my s in c e r e thanks to P r o f .

Ather H. S i dd i q i to uhom I am indebted f o r h is

s u p e r v i s i o n , guidance, i n va luab l e sugges t i ons ,

c o n s t r u c t i v e c r i t i c i s m and encouragement throughout

t h i s uork.

I am thank fu l to P r o f . S.i^. Alam f o r p r o v i d ing

me necessary f a c i l i t i e s in the Department of Zoo logy .

I am g r a t e f u l to Dr. Ni tya Nand, D i r e c t o r ,

Cen t r a l Drug Research I n s t i t u t e (CDRl ) , Lucknou, and to

Dr. A.B. Sen, Head, P a r a s i t o l o g y D i v i s i o n , f o r the use

o f research f a c i l i t i e s in the Cent ra l Drug Research

I n s t i t u t e .

The help g iven by Dr. Ran Govind, S c i e n t i s t ,

D i v i s i o n of P a r a s i t o l o g y , CDRI , i s g r a t e f u l l y acknouledged,

f o r he permit ted me to use a l l the f a c i l i t i e s a v a i l a b l e

in his l a b o r a t o r y , e s p e c i a l l y f o r the use of h is cu l ture

room and f o r p r o v i d ing a l l chemica l l y de f ined cu l tu re

media and f o r g i v i n g va luab l e sugges t i ons .

( SHAHIMAZ BAIMO )

N T E N T S

Page No.

I . INTRODUCTINN

I I . HISTORICAL R E U I E U

I I I . STATEI^ENT GF PRGBLEN

L\J. MATERIALS AND HETHOOS

\J, RESULTS AND DISCUSSION

1. Morphology

( a ) L i gh t microscopy

( b ) S t e r eoscan s t u d i e s

2. H i s t o l o gy . . .

3. H i s t o chemis t r y . . .

4. In v i t r o c u l t i v a t i o n

\LL, REFERENCES

\YIL. ABBREVIATIONS

W i n . SUr HARY

1 - 7

8 -49

50 -53

54 -77

78-104

105-110

111-135

136-165

165-173

174-222

223-225

226-228

* * * *

INTRODUCTION

Among the nematodes uhich inc lude a uery l a r g e

number of worms be long ing to d i f f e r e n t groups, the

f i l a r i i d s are of g rea t medica l and v e t e r i n a r y importance.

They cause much human s u f f e r i n g in many par ts o f the u o r l d ,

e s p e c i a l l y in the deve l op ing c oun t r i e s . In e a r l y s tages

o f i n f e c t i o n the pa t i en t s u f f e r s from c h i l l s , f e v e r , aches

and g ene ra l ma la ise . Th i s , i f n e g l e c t e d , r e s u l t s in

in f lammat ion of lymph glands ( l ymphaden i t i s ) and lymph

channels ( l y m p h a n g i t i s ) , thereby s t a r t i n g a l l e r g i c

r e a c t i o n s ending in e l e p h a n t i a s i s . The l a t t e r i s

c h a r a c t e r i s e d by enlargement of c e r t a i n par ts o f the body

due to the depos i t i on of e x t r a c e l l u l a r p r o t e i n , f i b r o s i s

and nec ros i s of t i s s u e s , causing phys i ca l d i scomfor t as

uG l l as d i s a b i l i t i e s r e s u l t i n g in much mental agony.

Not only t h a t , these f i l a r i a l worms cause t r o p i c a l

e o s i n o p h i l i a , i f a c c i d e n t a l l y , man gets i n f e c t e d u i th

f i l a r i a e of animal o r i g i n uhich cannot deve lop

s a t i s f a c t o r i l y in man. These f i l a r i a l worms may cause

d i s turbances in eyes and c e n t r a l nervous system a l s o .

The most common human f i l a r i i d s are Uucherer ia

b a n c r p f t i and Bruq5 a malay i . U. b a n c r o f t i i s p r e va l en t

in warm and humid coun t r i e s l i k e I n d i a , A f r i c a , China,

A rab ia , Nalaya, Formosa and West I n d i e s ; uhereas B, malayi

i s found in I n d i a , na laya , Southeastern Asia and East

I n d i e s e t c . These are the worms commonly r e spons ib l e f o r

l ymphang i t i s , lymphadeni t is and e l e p h a n t i a s i s . The other

f i l a r i i d s i n f e c t i n g man are Onchocerca uolv/ulus,

Dipetalonema pers tans , D. s t r e p t o c e r c a , l^ansonella o z z a r d i ,

Loa loa and D i r o f i l a r i a spp.

Ivulus occurs in Plexico, Guatemala, Sa l vador ,

Northuest Venezue la , A f r i c a and Yemen. I t causes

o n c h o c e r c i a s i s , r e s u l t i n g in deuelopment of f i b r o u s nodules

in the sk in . Dipetalonema perstans i s Found in Congo,

Uganda, South America and northern Argent ina . D. s t r e p t o c e r c a

i s found in Liest and Cent ra l A f r i c a . No e v i d en t symptoms

are produced except a p e r s i s t e n t headache, drousyness,

skin rash and eos inophi l i a. Plansone 11a o z za rd i i s common

in West I n d i e s , Yucatan, Panama, South America and in

nor thern Arqen t i a . The uorms are found in mesentr ies and

v i s c e r a l f a t . No pathogen ic symptoms are produced,

i n f e c t i o n r e s u l t s in an increase of blood e o s i n o p h i l s .

Loa loa i s common in Uest and Cent ra l A f r i c a . Adults

l i v e in sub-cutaneous t i s sue of man and make excurs ions

from place to p lace under skin causing i t c h i n g and c reep ing

s ensa t i on . They shou a s p e c i a l p r e f e r ence f o r c reep ing

in and about the e yes . I t causes pa in l e s s or i t c h y

edematous s u e l l i n g s c a l l e d "Ca labar s u e l l i n g s " which

appear suddenly , l a s t a feu days and then d i sappear , t o

reappear l a t e r someuhere e l s e . These are due to the

a l l e r g i c r e a c t i o n s , to metabo l i c products of the uorms or

t o p r o t e i n s l i b e r a t e d from in ju r ed or exp i r ed uorms.

f l i c r o f - i l a r i a loa may cause f a t a l e n c e p h a l i t i s , uhen they

pene t ra t e i n t o the brain and s p i n a l cord .

Gne spec i e s of D i r o f i l a r i a , 0. conJuct ivae has

been found to p a r a s i t i s e man in Europe, I n d i a , U .S .S .R.

and Tha i l and . I t l i v e s in cyst l i k e tumors of e y e , nose,

arm and mesentry.

The f i l a r i a l p a r a s i t e s of domest ic animals be long

to the spec i e s of Brugia , Dipetalonema, Onchocerca,

S e t a r i a , S t e p h a n o f i l a r i a , P a r a f i l a r j a and D i r o f i l a r i a .

The spec i es of Brugia p a r a s i t i s i n g cats and dogs

a r e , B, malayi in Ind i a ( O r i s s a ) , P'lalaya and Kenya coast

of A f r i c a ; B. pahanqi in Malaya, and B. pa t e i in Pate

i s l a n d and Kenya. They do not produce any pathogenic

symptoms. Only one spec i es of Dipeta lonema, D. reconditum

i s a very common pa ras i t e of dogs in U.S .A . but i t does

not produce any pathogenic symptoms.

The spec i e s of Onchocerca i n f e c t i n g domest ic

animals a re , £ . q ibson i p a r a s i t i c in c a t t l e . ^ I t i n j u r e s

h ides and carcasses by forming hard nodules , 0. i nd i ca

and 0. gut te rpsa i n f e c t c a t t l e and they a l so form skin

nodules . 0. r e t i c u l a t a (O. c e r v i c a l i s ) i nhab i t s the neck

l igament of horses in U.S.A. causing " p o l l i l l " and

f i s t u l o u s w i t h e r s , the m i c r o f i l a r i a e cause papular i t c h i n g

skin s o r e s . 0. a rm i l l a tus causes aneurysms in aor ta of

c a t t l e in A f r i c a ,

The spec i es of S e t a r i a p a r a s i t i c in domestic

animals a r e , 5. equina in horses , 5. l a b i a t o - p a p i l l o s a ,

5. c e r v i and d i g i t a t a in c a t t l e in I n d i a . They do not

cause any damage, but in n o n - s p e c i f i c hos ts , they are

c a r r i e d even to the brain and there i s a danger of invas i on

of c e n t r a l nervous system of sheep, goat and horses e t c .

The ' e p i z o o t i c c e r e b r o - s p i n a l nematod ias i s ' i s found to be

caused by immature S. c e r v i , bes ides t h i s , lumbar p a r a l y s i s

or 'Kumri ' has a l so been caused.

The spec i es of S t e p h a n o f i l a r i a p a r a s i t i s i n g c a t t l e

are S. assamensis causing humpsore in c a t t l e in Assam,

Benga l , Bihar and Or i s sa , z ahee r i causing e a r - s o r e in

Hyderabad and s t e l e s i causing skin and humpsore in

c a t t l e , goat and p i g in I nd i a .

The spec i es of P a r a f i l a r i a i n f e c t i n g domest ic

animals are P. bpv i co l a in c a t t l e and P. m u l t i p a p i l l o s a

in horses in old uo r l d . The uorms p i e r c e the sk in to

d epos i t embryos, causing "summer b l e ed ing " from smal l

"Modules and i n j u r i n g the h ides .

The spec i es of D i r o f i l a r i a i n f e c t i n g domest ic

animals are D. scao i ceps p a r a s i t i c in r a b b i t s , D. immit is

and D. repens p a r a s i t i c in dogs, in a l l uarm c l imates

and southern United S t a t e s . The dogs show r e s p i r a t o r y

d i f f i c u l t i e s .

The f i l a r i i d s i n f e c t i n g w i ld animals are spec i e s

o f Brug ia , Loa, Dipeta lonema, Onchocerca and D i r o f i l a r i a .

Tuo spec i es of Erugia are p a r a s i t i c in u i l d

animals . B. ma lay i , in t i g e r s , s lou l o r i s and an t - ea t e ra

in I n d i a , Halaya, Or issa and Kenya and B. pahangi in

monkeys, slow l o r i s , t i g e r s , other u i l d f e l i n e s and

a n t - e a t e r s in Plalaya.

Loa loa i s found in monkeys in Be lg ian Congo.

One spec i es of Dipetalonema, D. perstans i s a

common pa ras i t e of apes in ra in f o r e s t s of Uest and

Cen t r a l A f r i c a and in chimpanzee in Be lg ian Congo. Spec ies

of Onchocerca are found p a r a s i t i c in ante lopes and one

s p e c i e s of D i r o f i l a r i a , D. tennuis i s found to i n f e c t

r accoons.

The s p e c i a t i o n in p a r a s i t i c nematodes i s usua l l y

based on morphologi c a l charac te rs of adul ts but in f i l a r i a l

p a r a s i t e s , adult i s hard to f i nd and the only a v a i l a b l e

s tage i s m i c r o f i l a r i a e , o f t e n present in blood smears

made at n i gh t . The d iagnos i s o f most human cases of

f i l a r i a s i s i s based on d e t e c t i o n of m i c r o f i l a r i a e in

p e r i p h e r a l b lood . Conseguently attempts are made by

uorkers t o f i n d r e l a t i v e l y simple and r e l i a b l e methods of

d i f f e r e n t i a t i n g the spec i e s of f i l a r i a l uorms by

i d e n t i f y i n g m i c r o f i l a r i a e present in p e r i p h e r a l b lood .

6

r i o r p h o l o g i c a l l y , the c u t i c u l a r s t ruc tu res l i k e

c e p h a l i c p a p i l l a e , genera l body sur face and caudal

p a p i l l a e are of g rea t ualue f o r d i s t i n g u i s h i n g the

d i f f e r e n t f i l a r i a l i n f e c t i o n s . In m i c r o f i l a r i a e usua l ly

the nuclear charac te rs and presence or absence of sheath

i s taken i n t o c ons i d e ra t i on f o r i d e n t i f y i n g the genus

and spec i es o f f i l a r i a l uorms. Uhen these charac te rs are

not s u f f i c i e n t enough, the c epha l i c s t ruc tu res of

m i c r o f i l a r i a e (hook and s p i n e s ) , the pharyngeal thread

and Innenkorper prov ide good taxonomic t o o l s .

The h i s t o l o g i c a l f e a tu r e s l i k e musculature, nuclear

constancy of oesophagus and c e l l s of i n t e s t i n e are of

g r ea t value in taxonomy. I f the change in h i s t o l o g i c a l

s t ruc tu r e s i s s tud ied a f t e r treatment u i th c e r t a i n drugs,

i t may g i ve an important c lue f o r t reatment of f i l a r i a s i s ,

f o r example the drugs may form plugs over o r i f i c e s , they

may deve lop s t e r i l i t y in uorms, e t c .

The h i s tochemica l t e s t s nrov ide a neu taxonomic

t o o l f o r d i s t i n g u i s h i n g the spec i e s of f i l a r i a l worms by

l o c a l i z i n g the phosphatases, sulphatases and e s t e r a s e s

in m i c r o f i l a r i a e . These are very - spec i f i c in t h e i r

d i s t r i b u t i o n in d i f f e r e n t s p e c i e s .

The i j i v i t r o s tud ies g i ve a chance f o r s tudy ing the

l i f e c y c l e s tages outs ide the body of v e c t o r and e ve r y

d e t a i l can be s tud ied without i n t e r f e r e n c e u i th host t i s s u e .

I n v i t r o s tud i es orov/ide 3 chance to t e s t the e f f e c t of

neu drugs on p a r a s i t e on l y . Later the t o x i c i t y t e s t s

can be made in exppr imenta l animals as the nsu drugs can

never be t e s t e d on human sub j ec t s d i r e c t l y .

The techniques f o r d iagnos ing human f i l a r i a s i s and

c l a s s i f y i n g them in t o d i f f e r e n t qrouos are not very

advanced. The morpho l og i ca l , h i s t o l o g i c a l and

h i s t o chemica l t e s t s f o r d i f f e r e n t spec i es are not very

advanced, and the s tud i es on ijn v i t r o c u l t i v a t i o n are

a l s o qu i t e s c a t t e r e d ?nd inadequate . The crou f i l a r i a ,

Chandlere11a haukinqi found in the r i g h t a u r i c l e o f

hear t o f Corvus macrorhynchos ( U a g l e r ) , p rov ides a ve ry

good oppor tun i ty f o r a l l such s t u d i e s . Hence t h i s uiorm

has been chosen as a model snKcies f o r a l l above mentioned

s t u d i e s , as i t i s homologous with human f i l a r i a .

8

HISTORICAL REWIEU

1. (Morphology

( a ) L ight microscopy

( i ) Adult

The genus Chandlere l la was proposed by Yorke

and Maplestone (1926) f o r P i l a r j a bosei Chandler, 1924,

uhich uas descr ibed from racket t a i l e d rjronqo

(Oissemurus parad i scus ) , Cory us crone , P ica p ica and

Certh ia f a m i l i a r j s from the Z o o l o g i c a l Gardens,

Ca l cu t ta . Later Pand i t , f^enon and I y e r (1929) a lso

descr ibed th i s species from r i gh t a u r i c l e o f a emu in

rnadras. G i lbe r t (193D) descr ibed a second spec ies

C. s tan tch insky i from India uhich uas a lso descr ibed

by Sultana (1962) from the heart of a uhite backed

munia (Uroloncha s t r i a t a s t r i a t a ) . Li (1933)

descr ibed C. s inens i s from lungs and trachea of

Corpus s inens i s an'H Urocissa sinens is in China.

Tubangui and f^iasalungan (193?) descr ibed

C. lepidoqrammi from P h i l i p p i n e s from L^pidogramnius

cumingi. Tr lo f f ( 1 ) descr ibed £ . l i e n a l i s from

fxussia from horse. A l i (1957) recorded C. s inens i s

and added one more spec ies C. s ingh i f n m the heart

9

o f Corvus macrorhynchTS in Hyderabad. Yeh (1957)

d e s c r i b e d a neu s n e c i e s C. braz i l i e n s i s from green

b i l l e d touchsn frnrr B r a z i l . Rasheed (1960) r e p o r t e d

C. Co lumb iqa l l i nae fAunus t ine , 19'37) from Crncnpus

phaen icopte rc i s , and d e s c r i b e d ine neu s p e c i e s

C. t hapa r i from 3 c x i c o l a t o rqua ta from Hyderabad.

3ones (1961) d e s c r i b e d f l e x i u a q i n a I j s from

Coruus brachyrhynchos from nontqomery c oun t r y , Qhio.

Sul tana (1962) added t h r e e more spc5cies t o t h i s qenus

C. h ima layans is from Himalayan k e s t r e l (Ce rchne i s

t innuncu lus i n t e r s t i n r t u s ) , C. b u c k l e v i from y e l l o u

f r o n t e d p ied uoorioecker ( L e i o p i r u s mahra t t ens i s

b lan for r i i " ! and C. a ] i i f n m l a r g e p i ed w a g t a i l

( H o t a c i l l a maderaspa tons i s ) from Hyderabad.

C h a t t e r j e e , Son and Bhat tacharya (1965) d e s c r i b ed the

n r esen t s p e c i e s Chand le re lJa haukinqi from hear t o f

Corvus mocrorhynchos from Luckoou.

' i i ) ' M i c r o f i l a r i a

Since the adu l t f i l a r i a l uorms are d i f f i c u l t t i

o b t a i n the s p e c i a t i o n or d i a g n o s i s o f these uorms i s

p o s s i b l e on ly by c h a r a c t e r i z i n g and d i s t i n g u i s h i n g

m i c r o f i l a r i a e of d i f f e r e n t s p e c i e s p r esen t in

o s r i p h u r a l b l ood .

10

Ful lcborn (1Q13) 3tu'^iod the morpho loq ica l

d i f P e r e n c o s in d i f f e r u n t t n i cn f i l a r i a e . Feng (1933)

made a comparatiwe study of the m i c r o f i l a r i a e of

Brugia malayi and UuchBroria h a n c r o f t i and found the

m i c r o f i l a r i a e of 8. malayi to be t w i s t e d in appearance.

I yengar (1939) a l so s tud ied the d i f f e r e n c e s in

morphology of m i c r o f i l a r i a e of U. b a n c r o f t i and

n. malayi and found a c r i n k l e d appearance and uauy

contours in B. ma lay i . Ui lson (1956) gave a Feu

more charac te rs of m i c r o f i l a r i a e f o r d i s t i n q u i s h i n q

these tuo s p e c i e s . The m i c r o f i l a r i a e of B. malayi

are shor t e r than those of U. b a n c r o f t i and are

usua l l y tw i s t ed u i th curv/es and kinks, whereas

m i c r o f i l a r i a e of U. b a n c r o f t i are l onqe r , smooth and

wi th few curuos. The nuc l e i in 'n icrof i l a r i ac of

E. malayi are smudqed and ove r l app ing but they are

d i s c r e t e in U. b a n c r o f t i . T h e r j are two n u c l e i in

t a i l of malayi which arc absent in U. h a n c r o f t i .

Chandler and l\uari (1960 ) , in t h e i r t e x t book

' I n t r o d u c t i o n to P a r a s i t o l o g y ' , i d e n t i f i e d UuchDreria

h a n c r o f t i , Druqia ma lay i , Loa l o a , tJ ineta lonrma

ne r s tans , Plansonella o z z a r d i . Onchocerca uoluulus

and D i r o f i l a r i a i ^ n i t i s by d i s t i n g u i s h i n g thri ir

m i c r o f i l a r i a e on the bas is of orescncc or absence

11

of sheath and d i f f e r r . n cos in nuclear land marks.

Ro th^te in and Broun (1960) separated d i f f e r e n t

m i c r o f i l a r i a e on the bas is of nuclear cha rac t e r s , using

v/ital s t a i n s . Tay l o r (1950) s tud ied .the s t ruc ture of

m i c r o f i l a r i a e of Loa l oa , Uuchereria b a n c r o f t i ,

O i r p f i l a r i a immi t i s , D. rspens and D. ae th i ops .

SchPcher (1962) s tud ied in d e t a i l the s t ruc ture

of m i c r o f i l a r i a of Brugia pahangi and descr ibed a feu

p o s s i b l e f e a t u r e s f o r i t s rap id d i f f e r e n t i a t i o n from

m i c r o f i l a r i a e of B. malayi and B. p a t e i . Schacher,

Geddaui and Church i l l (1967) s tudied the nuclear

number of m i c r o f i l a r i a e and proposed t h e i r number as

a t e s t f o r i n t r a s p e c i f i c groupings and e v o l u t i o n in

Uucherer ia b a n c r o f t i found in d i f f e r e n t geograoh ic

a reas . Schacher and Geddaui (1969) again presented

an ana l y s i s of s p e c i a t i o n and e v o l u t i o n in Uucherer ia

b a n c r o f t i by the study of nuclear constancy ( e u t e l y )

in m i c r o f i l a r i a e .

Pnartinnz Baez (1176) descr ibed the m i c r o f i l a r i a

o f Inchocurca v o l v u l u s . He gave s p c c i a l a t t . jn t i on to

c e p h a l i c and caudal e x t r e m i t i e s and to some of the

c e l l s in major part nf the body. He recommended

s i m i l a r s tud ies in other endemic aroas so as t o search

the poss i b l e d i f ferenc-.s ,

12

Rpqprding c epha l i c and pharyngeal s t ruc tu r e s

and Innonkorper , uery l i t t l e unrk has been done only

in r ecent years .

As fi^^rly as 1966, Sivanandam and Frednr icks

d i s t i n g u i s h e d the m i c r o f i l a r i a e of Brugia pahangi and

B. malayi on the bas is of Innenkorper . Laurence and

Simpson (1968) s tud ied the c epha l i c hook and sp ines

o f m i c r o f i l a r i a e of Brngia , lu'uchereria, Loa,

C a r d i o f i l a r j a Onchocerca, Mansonel la, Uipotalonoma and

L i tomoso ides and found these charac t e rs to be very

s p e c i f i c f o r d i f f e r e n t oenera. Laurence and Simpson

(1969) again s ta ined c epha l i c s t r u c t u r e s , o r a l r i n g ,

pharyngea l thread , Innenkorper and some other important

s t ruc tu r e s l i k e e x c r e t o r y pore , pores of Schuanzgebi Ide

in m i c r o f i l a r i a e of L/uchereria, Brugia , Loa and

C a r d i o f i ] a r j a . Again Laurencc and Simpson ( I 9 7 l )

desc r ibed a l l the s t ruc tu res present in m i c r o f i l a r i a

o f Brug ia , the sheath, the c u t i c l e , the c e p h a l i c space,

the nuclear colur^^n, the sub - cu t i cu l a r c e l l s , the nerv f

r i n g , the e x c r e t o r y pore , the nharyngeal th read , the

Innenkorper , the c e l l , the c e l l s , the anal

v e s i c l e , the SchuanzgobiIde and the t a i l n u c l e i .

Reaarding tuo forms of m i c r o f i l a r i a e , KorkL

( 1 929) descr ibed the frorpholoqy of t y p i r a l and a t y p i c a l

13

forms of m i c r o f i l a r i a e in Uucherer ja b a n c r p f t i . He

(192n) addud that a t y p i c a l forms were much shortpr

and s l i g h t l y t h i c k o r . The length of c epha l i c space

was s h o r t e r , the nuc l e i o v e r l ap , the nerv/e r i ng

s l i q h t l y a n t e r i o r l y p laced and t a i l i s abrupt ly drawn

ou t . Schacher (1962) working on B. pahangi and

B. malayi showed that the body length of m i c r o f i l a r i a e

i s h i gh ly v a r i a b l e and hence cannot be taken as

c r i t e r i a f o r spec i e s d i a g n o s i s .

14

( b ) Stereoscan study

( i ) Adult

Although mic ro topog raph i ca l f e a t u r e s o f the head

r eg i on and the sur face s t ruc ture o f body c u t i c l e hav/e a

g r ea t s i g n i f i c a n c e in nematode taxonomy, yet v e ry

l i t t l e uork has been done to study the f i n e r d e t a i l s of

these s t ruc tu res by s te reoscan s t u d i e s .

A l l i s o n , Ube laker , Uebster and R idd le (1972)

desc r ibed a s i m p l i f i e d technique f o r p repara t i on of

helminths f o r scanning e l e c t r o n microscopy. Tf^isystated

tha t the specimens have t o be t r e a t e d in a manner f o r

making them s e l f suppor t ing in high vacuum and f o r

t h e i r su r f aces to car ry o f f any charge that b i i i lds up

due to e l e c t r o n bombardment. In t h i s uorkthey s tud ied

the a n t e r i o r r eg ion of Su l casca r i s sulcatum.

Dick (1972) s tud ied the u l t r a s t ruc ture of

c u t i c l e of Syphacia obye l a t a . Ueise (1973) s tud ied the

su r f a c e topography of c u t i c l e , a n t e r i o r and caudal ends

and the male and female r ep roduc t i v e system of

^'Temonchus contor tus by scanning e l e c t r o n microscopy.

Dick ^nd Uright (1974a) s tud ied the u l t r a s t r u c t u r o of

the c u t i c l e a s soc i a t ed u i th male r ep roduc t i v e s t ruc tu r e s

in nematode Syphacia o b v e l a t a . They found v a r i a t i o n s

in the c u t i c l e of v e n t r a l copu la to ry s t r u c t u r e s ,

c l o a c d p a p i l l a e , c l o ' i ca , s p i c u l e s , sp i cu l a r sheath,

gubern^culum and other accessory s t r u c t u r e s .

15

Dick and Wright ( i g74b ) i n a separstB papor

s tud i ed the c u t i c u l a r v a r i a t i o n s assoc i a t ed u i t h

e x c r e t o r y pore , vu lva and vagina v e ra . A r i z ono ,

natsuo and Yoshida (1976) did scanning o l e c t r o n

microscopy of S t r o n g y l o i d e s p l an i c eps . They s tud ied

the e x t e r n a l f e a t u r e s of p a r a s i t i c and f r e e l i v i n g

adul t f ema l es , f r e e l i v i n g males, i n f e c t i v e and f i r s t

s tage rhabd i t o i d l a r v a e . Hogger, Es tey , K i s i e l and

Zuckerman (1976) s tud ied the d i f f e r e n c e s in c u t i c u l a r

markings betueen young and o ld females of

Caenorhabd i t i s b r i g q sae . Kikuchi (1976) in h i s three

separa te papers s tud i ed c u t i c u l a r morphology of

A s ca r i s lumbr ico ides and compared i t u i th A. suum,

the c u t i c u l a r morphology of Asca r i d i a columbae and r-ffi •

the c u t i c u l a r s t ruc tu r e s of Terranova dec ip i ens and

Gnothostoma d o l o r e s i . Lopez C aba H e r o and Cast an ys

Cue l l o (1 976) s tud ied the f i n e r d e t a i l s o f c epha l i c

e x t r em i t y of S p l e n d i d o f i l ^ r i a mavis ( L e i p e r )

C^nnert, 1937 u i th s toreoscan e l e c t r o n microscope .

Setasuban (1976) s tud ied the c u t i c u l a r d e t a i l s of

Bathmostomum sangor i Cobbold, 1879 under s t e r eoscon

e l e c t r o n microscope. Uang and F u j i t a (1976) compared

the e x t e r n a l morphology of Ascar i s suum and

A. lumbr ico ides u i th scanning e l e c t r o n microscopy and

found no d i f f e r e n c e in the e x t e r n a l morphology of these

tuo .

16

Barus, K o t r l a and Tenora (1977) d id scanning

e l e c t r o n microscopy of s p i c u l a r sheath of some

t r i c h u r i d s . Shava and Leuis (1977) s tud i ed the

c u t i c l e o f oxyur id nematodes be l ong ing to genus Syphac ia .

Shoho and Uni (1977) s tud i ed the e x t e r n a l

f e a t u r e s of S e t a r i a d i g i t a t a ^ S, m a r s h a l l i , S.m. pande i ,

equina and 3. l a b i a t o - p a p i l l o s a under s t e r eoscan

e l e c t r o n microscope and a m p l i f i e d the d e s c r i p t i o n of

these spe c i e s in the l i g h t of s t e r eoscan s t u d i e s .

They a l s o d i scussed taxonomic importance of

these c h a r a c t e r s . Uertheim and Chabaud (1977) s tud i ed

the c e p h a l i c cha rac t e r s o f the e n t i r e f a m i l y

Pneumospirur idae ( T h e l o z i o i d e a - Nematoda) and in the

l i g h t o f these c h a r a c t e r s , they r e v i s e d the e n t i r e

f a m i l y .

r^archiondo and Sauyer (1978) made a s t e r e o s c a n

study of m i c r o t o p o q r a p h i c a l f e a t u r e s o f head r e g i o n of

Physa l op t e r a f e l i d i s . Tenora, U iger and Barus (1Q78)

d id scanning e l e c t r o n m i c roscop i c s t u d i e s on three

s p e c i e s o f the genus Syphac ia , S. o b v e l a t a , 3. n i qo r i ana

and 3. stroma. They po in ted out s i m i l a r t r a n s v e r s e

s t r i a t i o n s on the body but d i f f e r e n c e in the s t r u c t u r e

of htjad. U i g c r , Barus and Tenora (1 978) s tud i ed the

u l t r a s t r u c t u r e of head and sur f a ce s t r u c t u r e of the

c u t i c l e of Syphacia p e t r u s e u i z i , S. n i g e r i a n a ,

3. f r e d e r i c i and S. stroma. They d i s t i n g u i s h e d these

17

s p e c i e s from each other on head and c u t i c u l a r

s t r u c t u r e s . Uong and Brummer (1978) s tud ied the

c u t i c u l a r morphology of f i v e spec i e s of D i r o f i l a r i a ,

D. immi t i s , 0. corynodes , D. magnilarvatum, D. repens

and D. t ennuis . They a lso observed v a r i a t i o n s in

pa t t e rns on e n - f a c e view of head and v a r i a t i o n s in

the c u t i c u l a r pa t t e rns u i th the po r t i on and aspect

o f the uorm examined. Barus, U i g e r , Tenora and

Stanek (1979) observed the l i p d e n t i c l e s of Toxascar i s - •' ' mi

l e o n i n e , Toxocara canis and T. c a t i under s te reoscan

e l e c t r o n microscope,

( i i ) M i c r o f i l a r i a

Regarding the u l t r a s t r u c t u r e s of m i c r o f i l a r i a e ,

v e r y l i t t l e work has bee;n done. McLaren (1 972) s tud ied

the u l t r a s t r u c t u r e of the m i c r o f i l a r i a e of Dipctalonema

v i t o a L , D. se tar iosum, O i r o f i l a r i a immi t i s , Loa loa and

L i tomoso ides c a r i n i i . U 'Leary (1972) s tud ied the

u l t r a s t r u c t u r a l aspects of m i c r o f i l a r i a e of O i r o f i l a r i a

immit is as r econs t ruc ted from s e r i a l s e c t i o n s .

Tongu (1974) s tud ied the u l t r a s t r u c t u r e of

m i c r o f i l a r i a e of Brugia ma lay i . n i e q e v i l l c , M a r j o l e t

and Ucrmeil (1978) observed the m i c r o f i l a r i a e of

Dipetalonema viteac under SEM. Thoy found 2 types of

m i c r o f i l o r l a e in b lood of hamsters. These d i f f e r in

form of a n t e r i o r end. The l e ss numerous type has a

l o n g , f a i r l y thin (about 2 yum) neck made up of 12-14

r e g u l a r c u t i c u l a r annules. In other t ypo , neck i s

s ho r t e r and made up of 6-8 annules.

18

19

2 . H i s t o l o gy

Not much work on h i s t o l o g y of nematodes has been

done. Whatever has been done i s in Form of s c a t t e r e d

papers and con f ined to only a feu s p e c i e s . Schacher

(1962) s tud ied the h i s t o l o g y of d i f f e r e n t organs of

Brugia pahangi , a f i l a r i i d of c a t , at d i f f e r e n t

important l e v e l s . Later Ansari and Basir (1964) made

a comprehensiue study of h i s t o l o g i c a l c h a r a c t e r i s t i c s

of eve ry organ and system of Set a r i a c e r y j , a f i l a r i a l

p a r a s i t e of bui^'falo. Rest of the uorkers s tud ied the

h i s t o l o g y of i s o l a t e d par ts in some or the other uorm.

V/on S i ebo ld (1838) s tud ied the complex nature

o f the c u t i c l e in Ascar i s lumbr i co ides . A s i m i l a r

pa t t e rn was observed by de nann (1886) in the c u t i c l e

o f Enoplus communis. Goldschmidt (1906) desc r ibed

9 l a ye r s in the c u t i c l e of A s c a r i s . Mart in i (1912)

s tud ied the c u t i c l e o f Oxyuris and found 9 l a y e r s in

i t . Schacher (1962) d id not go i n t o the d e t a i l e d

s t ruc tu r e of the c u t i c l e of Brugia pahangi and he

s imply descr ibed tuo l a y e r s in i t , a c o r t i c a l d e l i c a t e

b a s o p h i l i c l aye r and a f i b r i l l a r r a f r a c t i l e e o s i n o p h i l i c

l a y e r . Ansari and Basir (1964) descr ibed 8 c u t i c u l a r

l a y e r s in S e t a r i a c e r v i . I n g l i s (1964) s tud ied the

s t ruc tu r e of nematode c u t i c l e . Asadov (1973) worked

on the c u t i c l e of T r i c h u r i s o v i s . He observed 6 l a y e r s

20

in the c u t i c l e . Magqnti (1979) s tud ied the c u t i c u l a r

s t r a t a in Enopli<? and proposed a system of nomenclature

based on c u t i c u l a r s t r a t a in nematodes as compared to

Enopl ia model.

Uieus d i f f e r r egard ing the o r i g i n of the c u t i c l o e

a l s o . n i j l l e r (1929) descr ibed i t as a s e c r e t i o n

product . Chitu/ood (1936) be l i eued that var ious l a y e r s

of the c u t i c l e deve lop as pro top lasmic condensat ion

from the l i v i n g c e l l . Ansari and Basir (1964) agree

u i th Chi tuood 's v i e u .

The c u t i c l e i s f o l l o w e d by s u b - c u t i c l e which i s

the middle l aye r o f the body u a l l . i t oua r t (1906)

c a l l e d i t ep ide rm is , uh i l e s tudy ing anatomy of

Oncholaimus. There are d i f f e r e n t v iews r egard ing i t s

o r i g i n . Hamann (1895) regards i t ec todermal whereas

zur j t r a s s e n (1904) b e l i e v e s i t t o bo mesodermal in

o r i g i n . Schacher (1962) did not go i n t o the d e t a i l s

o f the o r i g i n of t h i s l aye r and he s imply desc r ibed

i t s th i cken ings at d o r s a l , v e n t r a l and l a t e r a l chords.

Ansar i and Basir (1964) d i sag ree with the t e rmina logy

and o r i g i n of t h i s l aye r and c a l l e d i t s u b - c u t i c u l a .

The importance of musculature in nematodes was

f i r s t o f a l l emphasised by Schncider (1860 ) . He

sugges ted that thu form, number and arrangement of

muscle c e l l s are of fundamental imoortance in the

c l a s s i f i c a t i o n of nematodes. On the bas i s of these

21

c h a r a c t e r s ho proposed the tnrm p la tymyar ian and

coo lomyar ian . The nematodes having f i b r i l l a r p o r t i o n

o f c e l l s f l a t towards the body c a v i t y are p la t ymyar ian

and those having f i b r i l l a r p o r t i o n o f c e l l s bea r ing

g r o o v e , d i s t a l l y making the sa rcop lasmic par t t o d ip

doun i n t o and between the c o n t r a c t i l e l a y e r s , p resent

on e i t h e r s ide of the c e l l s are coe l omyar ian . Fur ther ,

he took i n t o c o n s i d e r a t i o n the number of muscle c e l l s

i n each s e c t o r , i f f e u , he c a l l e d i t meromyarian, and

i f numerous, po lymyar ian . He s t a t e d tha t meromyarian

forms are p l a t ymyar i an , and polymyar ian forms are

coe l omyar i an . S c h n e i d e r ' s uork l o s t importance when

(Martini (1916) found tha t po lymyar ian and coe lomyar ian

nematodes are meromyarian and p la tymyar ian in f i r s t

l a r v a l s t a g e . Th is proves tha t polymyary and

coe lomyary i s reached in l a t e r s tage o f deve lopment .

Schachcr (1962) d esc r i bed musculature of Bruqi a

pahangi to be coe l omyar i an , not s epara t ed by d o r s a l

and v e n t r a l chords . f^uscle c e l l s in g r a v i d f ema les

are compressed to th in dense l a y e r u i th n u c l e i among

f i b r i l l a r e l ements .

Ansar i and Bnsir (1964) d e s c r i b e d musculature

o f S e t a r i a c a r y i t o be po lymyar ian and coe lomyar ian

t y p e , broken at four p l^ccs by d o r s a l , v e n t r a l and tuo

l a t e r a l chords. They desc r i b ed s p e c i a l i s e d muscles

22

support ing oesophagus, i n t e s t i n e , uulva, anus and

SDicu les , Hirumi, Raski and Gones (1971) s tud ied the

morpho log ica l aspects of platymyarian and sha l l ou

coelomyarian muscles in Trichodorus c h r i s t j e i and

Longidorus e longatus (p lant nematodes). Bogoyav/lens k i i

and Skr jab in (1971) made comparative h i s t o l o g i c a l

i n v e s t i g a t i o n s of the somatic musculature of c e r t a i n

nematodes of the sub-order s p i r u r a t a .

Regarding the body f l u i d , Stewart (1906) s

descr ibed a j e l l y l i k e sut^tance having nuc le i in i t .

Schacher (1962) did not mention anything about body

f l u i d . Ansari and Basir (195A) descr ibed a granular

body f l u i d in Se t a r i a c e r v i . They suggested that t h i s

f l u i d i s necessary as c i r c u l a t o r y system i s absent

and th i s f l u i d may serve as haemocoelomic f l u i d of

o ther i n v e r t e b r a t e s .

The connect ive t i s sue in nematodes has been

descr ibed by Schneider (1902) under the term

" Bindegeuebe" . Looss (1 905) c a l l e d i t " s t rand l i k e

organs'" and Goldschmidt (1906) proposed the term

" I so la t i ongeuebe " .

Schacher (1962) uh i l e studying h i s t o l o g y of

Brugia pahangi did not mention anything about connect ive

t i s s u e . Ansari and Basir (1964) descr ibed nucleated

strands between oesophagus and muscle l aye r in

5. c e r v i .

23

Uork on h i s t o l o g y of oesophagus of nematodes i s

s c a t t e r e d . f^art in i (190S) and Chituood (1 930) s tud ied

the oesophagus of nematodes and descr ibed that the

oesophagus i s covered e x t e r n a l l y as w e l l as i n t e r n a l l y

by a membrane c a l l e d ' t un i ca p r o p r i a ' and regard i t

t o be the s e c r e t i o n of marg inal n u c l e i ,

Schacher (1962) descr ibed tuo oar t s of oesophagus,

the muscular and the g landular in B. pahangi ; the

muscular part i s deeply e o s i n o p h i l i c u i th l a rge nuc l e i

and t r i a n g u l a r lumen and the g landular part i s g ranu la r ,

vacuo la t ed u i th many gland and suppor t ing c e l l n u c l e i .

He descr ibed the presence of a b i l obed oesophago-

i n t e s t i n a l v a l v e , h i s t o l o g i c a l l y s i m i l a r to oesophagus.

Ansari and Basir (1964) a l so descr ibed tuo

pa r t s in oesophagus of 5. c e r v i , e x t e r n a l l y covered

by ' t un i ca pro .pr ia ' , u i th t r i r a d i a t e lumen, supported

by s p e c i a l muscles ca l lnd the somato-oesophageal

muscles. They a lso descr ibed o e s o p h a g o - i n t e s t i n a l

v a l v e , through uhich oesophagus opens i n t o the i n t e s t i n e .

Looss (1905) cons idered the o e s o p h a g o - i n t e s t i n a l va lue

as a product of d i f f e r e n t i a t i o n of i n t e s t i n e . Plagath

(1919) descr ibed i t as a s p e c i a l i s e d part of

oesophagus.

Chituood (1931) proposed the term oesophago-

i n t e s t i n a l v/alv/e. Onmori, Yoshimura and I sh igooka

(1976) did a comparat ive study of oesophageal lumen

24

in s t r o n q y l o i d e a . They presented the cross s e c t i o n s

o f oesophageal r eg i on of 13 spec i e s of Ancy los tomat idae ,

6 o f 3 t r o n g y l i d a e , one of Stephanuridae and § ' o f

T r i chonemat idae , and rccogn ised 5 types of oesophagea l

c u t i c u l a r l i n i n g s . They suggested that s t ruc ture of

oesophagus in cross s e c t i on a l l ows the d i f f e r e n t i a t i o n

of s p e c i e s . Sood and Seha jpa l (1979) s tud ied the

oesophagus of H. contor tus and found the lumen to be

l i n e d u i th c u t i c l e .

The h i s t o l o g y of i n t e s t i n e of nematodes has

a l s o not r e c e i v e d much a t t e n t i o n . Chituood (1930)

gave an i n t e r e s t i n g method f o r c l a s s i f y i n g nematodes

depending on the number, the form, the he ight and the

shape of c e l l s in cross s e c t i on and grouped the

nematodes in t o d i f f e r e n t c a t e g o r i e s . Those having

l a rge number of c a l l s are termed as myriocytous and

those having i r r e g u l a r lumen are an i socy tous .

Schacher (1962) descr ibed i n t e s t i n e to be made

up of a th in hya l ine outer membrane and a b a s o p h i l i c

granular ep i the l ium conta in ing 5-9 nuc l e i per s e c t i o n

at mid r e g i o n . Ansari and Basir (1964) whi le

d e s c r i b i n g h i s t o l o g y of S. c e r v i gave t h e i r op in ion

that t h i s e f f o r t of c l a s s i f i c a t i o n i s uneven and

t roub lesome. They descr ibed the i n t e s t i n e of c e r v i

t o be myriocytous and an i socy tous . They suggested

that myriocytous nematodes are always an i socy tous .

25

Bogolepova (1975) s tud ied the h i s t o l o g i c a l

c h a r a c t e r i s t i c s o f i n t e s t i n a l s t ruc tu r e of Arnidostomum

a n s e r i , Uncinar ia s tenocephala and Oesophagostomum

uenulosum. Bogoya\ylenskii (1976) s tud i ed the

h i s t o l o g y of f i n e s t ruc tu res o f nematodes. Sood and

Seha jpa l (1979) found i n t e s t i n a l ep i the l ium prouided

with deve loped brush borders in H_. c on to r tus .

Uieujs among workers d i f f e r r ega rd ing u a l l of

the i n t e s t i n e which c ons i s t s of a s i n g l e l ayer o f

e p i t h e l i a l c e l l s , prov/ided u i th conspicuous l i n i n g on

the i n t e r n a l sur face knoun as 'Stabchensaum' or

b a c i l l a r y l a y e r .

Daegersk ide Id (1894) found that b a c i l l a r y l aye r

known as 'Stabchensaum' i n t e r n a l l y and the outer

e p i t h e l i a l c e l l l a y e r o f the i n t e s t i n e c ons i s t s o f

long separate rods . Looss (1905) descr ibed

'Stabchensaum' as c u t i c u l a r l a y e r . Quack (1913) made

a d e t a i l e d study of the i n t e s t i n a l c e l l s and

concluded that the rods descr ibed by 3aege r sk i o e Id

(1B94) are l o n g i t u d i n a l s e r i e s o f alv/eoli and i n t e r -

a l v e o l a r substance. Hether ington (1923) s ta t ed that

s u b - b a c i l l a r y l aye r c ons i s t s of f i n e granules which

appear to be connected with each other and with the

rods which form the b a c i l l a r y l a y e r . I^ueller (1929)

suggested that b a c i l l a r y and s u b - b a c i l l a r y l a y e r s

26

c o n s t i t u t e neuromotor system in A s c a r i s . Chituood

(1930) exp la ined the nature of b a c i l l a r y l aye r and

gave three p o s s i b i l i t i e s .

1. B a c i l l a r y l a y e r i s a dewelopmont of minute

tubules uhich aid in absorp t ion or e x c r e t i o n .

2. I t i s a s e c r e t i o n product of p r o t e c t i v e

nature .

3. I t i s a l a y e r o f amalgamated degenerate

c i l i a .

Ansari and Basir (1964) descr ibed b a c i l l a r y

l a y e r c o n s i s t i n g of c i l i a l i k e rods . The rods are

usua l l y fused t o g e the r forming a membrane, but at

p l a ces they are s epa ra t e . They found t h i s l aye r to

be high in card iac part of i n t e s t i n e but lou in r e c t a l

p a r t . There, i s a l so a granular sub-baci l l a r y l a y e r or

' d e c k s c h i c h t ' cor responding to the l a y e r o f basa l

granules in c i l i a t e d ep i the l ium uhich i s f o l l o w e d by

e x t e r n a l pro top lasmic zone of i n t e s t i n e . They a lso

desc r ibed the basal lame l la p i e s en t outer to the

pro top lasmic zone.

Only Ansari and Basir (1964) desc r ibed an

i n t e s t i n o - r e c t a l va l v e surrounded by sph inc te r muscles.

They desc r ibed the rectum to be l i ned by c u t i c l e and

supported by s p e c i a l muscles, depressor ani and d i l a t o r

an i .

27

Uorkors l i k e Eberth (1363 ) , Hamann (1395 ) ,

Ehlers (1899) and V/oltzenloge 1 (1902) descr ibed

r e c t a l g lands. Looss (1905 ) , whi le s tudy ing

Ancylostoma concluded that these are made of connectik/e

t i s s u e . Mart in i (1913) a l so b e l i e v e d that these are

the r e c t a l l i gaments . flagath (1919) took them as

sarcoplasm of the sph inc te r muscles, as none of these

workers could see the duct from these s t ruc tu r e s

l e ad ing in t o rectum. Chituood (1930) uas f i r s t t o

observ/e the o r i f i c e s of r e c t a l glands in R h a b d i t i s ,

f^acrac is , Cephalobe l l u s and Hystr ingnathus. Baker

(1936) conf irmed Chi tuood 's obse r va t i ons in He t e rak i s .

Schacher (1962) did not mention anything about these

s t r u c t u r e s . Ansari and Basir (1964) though observed

these s t ruc tures in S e t a r i a c e r v i but could not

conclude t h e i r nature as they were unable to observe

any duct or any o r i f i c e from these s t r u c t u r e s . Only

Ansar i and Basir (1964) s tud ied the h i s t o l o g y of

the c l oaca j f male in S. c e r v i .

The h i s t o l o g i c a l anatomy of female r ep roduc t i v e

system was f i r s t s tud ied by Chitwood (1929) where he

desc r ibed o va r i e s t o begin with a cap c e l l , which

cove rs the b l ind end of ovary and a part o f o v a r i a l

e p i t h e l i u m . Musso (1930) gave a comple te l y d i f f e r e n t

v iew and regarded the cap c e l l t o be u n d i f f e r e n t i a t e d

germinal stem c o l l which gave r i s e both to e p i t h e l i a l

37

and germinal c u l l s . Schacher (1962) descr ibed tuo

T u a r i e s , op i s thode Iph i c with germinal and growth

zones and a t e rm ina l cap c e l l . In 3 e t a r i a ce ru i

Ansar i and Basir (1964) s t a t ed that ovary i s d i v i s i b l e

i n t o tuo zones , the germinal zone f o l l o w e d by growth

zone. The t i p of the germinal zone i s covered by the

a p i c a l c e l l .

The h i s t o l o g i c a l anatomy of o v iduc ts was f j r s t

s tud i ed by Schacher (1962) in Brugia pahangi. He

observed the ov iduc ts with narrow lumen surrounded

by th i ck cubo ida l ep i the l ium of about 6 c e l l s per

s e c t i o n . Later Ansari and Basir (1964) descr ibed

the h i s t o l o g y of o v iduc ts of S e t a r i a c e r v i , having

narrow lumen surrounded by cubo ida l c e l l s . Onushko

(1974) s tud ied the h i s t o l o g i c a l s t ruc tu r e of female

g e n i t a l organs of Syngamus t rachca at d i f f e r e n t s tages

o f post-embryonic development.

D i f f e r e n t workers put f o r t h d i f f e r e n t v iews

r ega rd ing ^hu o r i g i n of rcceptaculum semin is . "'lagath

(1919) b e l i e v e d i t to be part of o v i duc t . Chitwood

and Chitwood (1933) descr ibed i t as a part o f u terus .

Schacher (1962) stat(>d that the wa l l s of seminal

r e c e p t a c l e are th i ck made of high cubo ida l c e l l s . He

d id not mention i t s o r i g i n . Ansari and Basir (1964)

b e l i e v e that seminal r e c e p t a c l e i s a mod i f i ed par t of

u terus .

29

Schacher (1962) dDscribod the h i s t o l o g y of

uterus in B. oahangi where th ^ u a l l i s tuo l aye red

c o n s i s t i n g o f an outer musclo coat and an inner

ep i the l ium of t a l l c e l l s , Ansari and Basir (1964)

a l s o descr ibed the h i s t o l o g y of uterus of S e t a r i a ce ru i

where the u a l l s c o n s i s t of outer muscular coat o f

c i r c u l a r muscles and inner l aye r of l a r g e , columnar,

nuc l ea t e c e l l s .

Schacher (1962) descr ibed the h i s t o l o g y of

vag ina in B. pahangi. He d i v i d ed i t i n t o tuo r e g i o n s ,

vag ina u te r ina and vag ina vc/ra . Vagina u te r ina has a

t h i c k outer muscle coat and an e q u a l l y th i ck inner

l a y e r o f cubo ida l c o l l s whereas vag ina \je^Ta i s made

o f s e v e r a l l a y e r s of c i r c u l a r and ob l ique muscles.

Ansar i and Basir (1964) a l so descr ibed the vag ina of

S e t a r i a c e r v i having the same c h a r a c t e r i s t i c s . The

d e t a i l s of vu lva have been descr ibed f o r the f i r s t

t ime by Ansari and Basir (19G4) in S e t a r i a c c r v i .

5ood and Knur (1977) s tud ied the h i s t o l o g y of vulva of

Haemonchus conto r tus . They found an outer c o l o u r l e s s

and innur th i ck s c l e r o t i s u d l aye r with smal l

p ro top lasmic co re . The e x t r a s w e l l i n g s are formed

by c u t i c l e .

Mo other worker j j r l i e r than Schacher (1962)

s tud i ed the h i s t o l o g i c a l d e t a i l s of male r ep roduc t i v e

30

system. HG s ta tod TNO tubular s o l i d t e s t i s u i th two

zones , the qGrminal and th^ growth zone in Brugia

pahangi . He observ/f-d thy t e s t i s s t a r t i n g as a s i n g l e

c c l l , f o l l o u c d by a suminal v u s i c l e u i th wa l l s o f

v a r i a b l e th i ckness . I t i s b a s o p h i l i c and leads in t o

the vas de f e rens u i th ten nuc l e i per s e c t i o n . The

vas de f e rens i s f o l l o w e d by a muscular e j a c u l a t o r y

duct which opens outs ide the body through c l oaca .

Ansari and Basir (1964) descr ibed s i m i l a r c h a r a c t e r i s t i c s

o f male r ep roduc t i v e system in Set a r i a c e r y i .

Schacher (1962) dcscr ibcd the capitulum of l e f t

s p i c u l e to bo tubular and r i g h t s p i c u l e beg inning with

t h i c k hol low tube with a med ia l l y d i r e c t e d a l a , the

lumen i s g radua l l y s h i f t e d and reduced to form smal l

channel in B. pahangi . Ansari and Bas ir (1964)

desc r ibed two unequal and d i s s i m i l a r sp i cu l e s in

S e t a r i a c e r v i . The l e f t sp i cu l e i s l a r g e r than r i g h t ,

having long p i t t e d sha f t f o l l o w e d by t a p e r i n g b l ade .

Tht. blade i s f l anged assuming gu t t e r shape. The

r i g h t sp i cu l e i s s t o u t , curved and boat shaped. They

desc r ibed thu s o i c u l e s t o be covered by a c u t i c u l a r

l a y e r h .v inq a pro top lasmic co re .

a i

3. H is tochemist ry

( a ) Acid phosphatase

As e a r l y as 19^6, Chourihury, Dasqunta, Ray and

Bhaduri s tudied the h is tochet i i ca 1 pa t t e rn of m i c r o f i l a r i a

o f Liuchereria b a n c r o f t i and found ac id phosphatase to

be absent in the Innenkbrper,

P i t i t h o r y (1T66) uas f i r s t to r epo r t the enzyme

a c t i v i t y d i f f e r e n c e s in m i c r ^ f i ] a r i a e j f d i f f e r e n t

s p e c i e s . This p rov ides a p o s s i b i l i t y of a neu taxonomic

t o o l f o r i d e n t i f y i n g the f i l a r i a l uorms.

The f e a s i b i l i t y of s epara t ing f i l a r i a l worms has

subsequent ly been deminstrated by Cha l i f oux and Hunt

( 1971 ) . These unrkers shiued s t r i k i n g l y d i f f e r e n t pa t t e rns

f o r ac id phosphatase a n t i u i t y in m i c r o f i l a r i a e of

O i r o f i l a r i a immit is and Dipetalonema reconditum even in

mixed i n f e c t i o n s .

Balbo and Abate ^1972) not only cnnfirmed the

obse r va t i ons of C h a l l f o j x and Hunt hut r epor ted a t h i r d

d i s t i n c t pa t t e rn f o r O i r i f i l a r i r ^ repens. Cha l i f oux ,

Hunt, Garc ia , Sehgal and Comiskey (1^73) went s t i l l

f u r t h e r f o r c l a s s i f y i n g the d i f f e r e n t f i l a r i a l uorms.

They combined ac id phosphatase a c t i v i t y u i th the

i n f o rma t i on on l e m t h and prencs of sheath to compose

a dichotomous key i d e n t i f y i n n 11 snec i es of m i c r o f i l a r i a e

from the neu world mnkeys .

32

Hedinqton, (^ontpDmery, Deruis and Hockmeyer (1975)

d i f f e r e n t i a t e d the m i c r o f i l a r i a e i f Bruqia pahangi and

s u b - p e r i o d i c B. ma l a y i by l o c a l i z i n q the acid phosphatase

ac t i v/ i t y .

Teruedou and Huff (1976) s tud ied the ac id

phosphatase a c t i v i t y in m i c r o f i l a r i a e of Uucherer ia

b a n c r o f t i . Braun-flunz i nqe r and Southgate (1977) s tud ied

the ac id phosphatase a c t i v i t y in m i c r o f i l a r i a e of

Onchocerca uo l vu lus , causing onchocercJas is in Uest A f r i c a ,

Four d i s t i n c t pa t t e rns of enzyme s t a i n i n g uere observed .

They concluded that a number of b i o l o g i c a l s t r a i n s or

v a r i a n t s of Hnchocprca vo l vu lus c o - e x i s t in Uest A f r i c a

and suggested f o r f u r t h e r research that could r e s u l t in

the p r a c t i c a l a p p l i c a t i o n in onchoce r c i a s i s c o n t r o l

programme. G'^ar (1977) s tud ied the d i s t r i b u t i o n of

a c i d phosphatase in l a r v a l s tages of IJuchereria b a n c r o f t i ,

Brugia ma lay i , B. pahangi and D i r o f i l a r i a immit is in

mosquito , and sunqested that an accurate d iagnos i s of

these g e n e r a could be made on the bas is of pa t t e rn of

d i s t r i b u t i o n . ']mar and Kuhlou (1977) d i f f n r e n t i a t e d

the m i c r o f i l a r i a e of Loa loa and iJinetalonema pcrstans

from Cnmeroun on the b f s i s of ncid phosphatase a c t i v i t y .

Omar (1978) performed hi s t T C h e i ca 1 d i f f e r e n t i a t i o n in

m i c r o f i l a r i a e of Inchocerca vo l vu lus by l o c a l i z i n g

ac id phosphatase, in IJn 5t A f r i c a n r a i n - f o r e s t s ( L i b e r i a )

33

Sudan-savana (Upper V o l t a ) , Guatemala and Yemen. He

d i s t i n g u i s h e d 5 d i s t i n c t s t a i n i n g pa t t e rns , Frequent ly

present in a s i n g l e i n f e c t e d person. Aaeinn, s t r a i n

d i f f e r e n c e or other f a c t o r s uere cons idered to be

causing these d i f f e r e n c e s , 'Irnar and Schulz-Key (1978)

s tud ied the ac id phosphatase a c t i v i t y in l a r v a l s tages

of Onchocerca volvyulus deve lop ing in the v/ector,

S imu lium dam no s urn. l/oen and Blotkamp (197 8) s tud ied

ac id phosphatase a c t i v i t y in m i c r o f i l a r i a e j f Oipntalonema,

D i r o f i l a r i a , Dnchocerca and S e t a r i a spp. Th.3y added that

s t a i n i n g pa t t e rn f o r ac id phosnhatase a c t i v i t y may hn Ip

in d i f f e r e n t i a t i o n of m i c r o f i l a r i a e of d i f f e r e n t spec i e s

w i th in the same host . Yen and f'iak (197R) l o c a l i z e d

the acid phosohatase a c t i v i t y in m i c r o f i l a r i a e of

Brug ia , Uucherer ia , 0 i r o f i l a r i a and B r e i n l i a . They

concluded that ac id ohosphatc^so a c t i v i t y in m i c r o f i l a r i a e

g i v e s s u f f i c i e n t l y c h a r a c t e r i 3 t i c and c ons i s t en t r e s u l t s

f o r di f f e re nt i a'".i 3n of oven c l o s e l y r e l a t e d snocius

l i k e Brugia malpyi and B. pahanni, O i r i f i l a r i a rcpens

and D, immit is and B r e i n l i a b o o l i a t i from B, s u r q e n t i .

The enzyme rUs t r i bu t i on of Plalaygian and ru ra l s t r a i n

of b'. b a n c r o f t i uos d i f f e r e n t f r m those of othtT

re g ions .

34

Not much a t t on t i on has beun paid to the enzyme

l o c a l i z a t i o n sf adult unrms. (âki, I t o and Yanagisaua

(1977) s tudied acid nhospbatase m adult D i r o f i l a r i a

immit i s . fâki and Yanaqisaua (lQgO) jbserund high acid

phosphatase a c t i v i t y in D i n f i l a r i a immit is at pH 3.Q-5.R.

The body u a l i and reproduct i ve organs showed high acid

phosphatase a c t i v i t y .

Among other nematodes Er]angur and Gorshon (1970)

s tud ied acid phosphatase a c t i v i t y in Turbatr ix a c e t i .

Rui tonbrrg and LoenHorsloot (1Q71) Found acid phosphatase

in i n t e s t i n a l t r a c t and brush borders of larvae of

Anisakis sp. fêznik ^ 9 7 1 ) l o c a l i z e d acid phosphatase

a c t i v i t y in the c u t i r l n of Ascar id ia q a l l i and Ascar is

lumbr ico ides . B o l l a , L 'e inste in and Lou (1974) worked

on the acid phosphatase a c t i v i t y in deve lop ing and

pqeing Nippostrongylus b r a s i l i e n s i s . They found nreates t

a c t i v i t y in th i rd staqo larvae and T-day p o s t - i n f e c t i o n

adu l t s . Seniuta (1975) l o c a l i z e d acid phosphatase in

deve lopmentt^l s tages of T r i c h i n c l l a s p i r a l i s . He

observed that a c t i v i t y var i ed uith embryonal, m i g ra t ing ,

deve lop ing muscular and invas ivn larvae and i n t e s t i n a l

uorms. Sharma, Singh and Sharna (1976) studied ac id

phosphatase in Stephanurus duntatus. They found maximum

a c t i v i t y at pH 4 .0 , in orsoohc.gu3, i n t e s t i n e , ovary ,

35

u t e r i n e u a l l , cqqs , c u t i c l e and in the t e s t i s . f'laki and

Yanagisaup (1930) obsRrv/ed hioh ac id phosphatase a c t i u i t y

at pH 4 . 5 - 6 . 0 in Ang ios t rongy lus cantonens is . Host of

the a c t i v i t y uas l o c a l i z e d in the body u a l l .

( b ) A l k a l i n e phosphatase

Choudhury, Dasgupta, Ray and Bhaduri (1956)

l o c a l i s e d a I k a l i n e phosphatase in m i c r o f i l a r i a e of

Uuchere r i a b a n c r o f t i . They found Tnnenkorper to be h igh ly

p o s i t i v e . A f e e b l e r e a c t i o n l'3s observed in the c u t i c l e

and n u c l e i . R oy ch ludhury, L a h i r i , Fenedle and Nabu ^ 975)

r epor t ed a lka l inp phosphatase c i c t i v i t y in the v e c t o r

Aedes aegyp t i a f t e r f e ed ing i t on a dog p o s i t i v e f o r

D i r o f i l a r i a repens. Those authors a l so s tud ied enzyme

a c t i v i t y in the adult uorms and found i t to bo g r ea t e r

in male than in f emale .

Ueen and Blotknmp f i g 7 3 ) s tud ied the a l k a l i n e

phosphatase a c t i v i t y in m i c n f i l n r i a e of Oipeta lonoma,

D i r o f i l a r i a , nnchocerca find Se t a r i a spp . , and suggested

tha t the pat terns of enzyme a c t i v i t y may aid to the

d i f f e r e n t i a t i o n of m i c r o f i l a r i a e of d i f f e r e n t spec i e s

u i t h i n the same host .

Among other nematodes, Segidn ( I 9 7 l ) l o c a l i z e d

a l k a l i n e phosphatase in nerve c e l l s of A s ca r i d i a g a l l i .

Seniuta (1975) s tud ied a l k a l i n e phosphatase in

deve lopmenta l s taqes of T r i c h i n o l l a s p i r a l i s and found

i t to be absent.

36

( c ) Adenosine trip'iosphafca'je

Only Couinriiiar, Cauande and Harinath (1 974)

de tec ted adinosine t r iphosphatase a c t i v i t y in homogenates

of n i c r o f i l a r i a e Liuchererid b a n c r o f t i . No other record

in f i l n r i a l uorms i s a v a i l a b l e .

Among other nematodes Pouyakel (116S) studied

adenosine t r iphosph?tase a c t i v i t y in muscle t i s sue

mitochondria of p ig Ascar i s . Ruitenberg and Loenders loot

( l 9 7 1 ) found adenosine t r iphosphatase in c u t i c l e of

Anisakis l a rvae . Reznik (1971) found AtPase in c u t i c l e

of both the spec ies v i z . , Ascar id ia g a l l i and Ascar is

lumbr ico ides . Bok (1973) studied d i s t r i b u t i o n of

adenosine t r iphosphatase in sorne hplminths. Hayashi (1973)

de tec ted adenosine t r iphosphatase f o rma t i i n by

mitochondria from Ascar is lumbricoides su i s . Pavlov

(1973) studied the r o l e of adenn3ine t r iphosphatase in

t ranspor t of amino-acids in -tscaris suwv. Van den Bossche

(1974) detec ted a low ,-mlecular c j n t en t of membrane

bound adenosine t r iphosphatase in mitochondria of

Ascar is suum muscles. Heznik (1975) found magnesium-

dependent adenosine t r iphosohatase in i n t e s t i n a l

ep i the l ium, medial nerve cord, somatic musculature,

oesophageal and reproduct ive system of Ascar id ia g a l l i .

Seniuta (1975) l o c a l i z e d adenosine t r iphosphatase in

37

developmental staqes of Tr ichine11a s p i r a l i s . He obsaryed

that the activ/ity va r i ed with embryonal, m ig ra t ing ,

deve lop ing muscular and inva^iue larvae and i n t e s t i n a l

worms. Ahn found adenosine tr inhosphatasn

a c t i v i t y in sub-cut i cu la r s y n c y t i a l c e l l s , s e r i a l zone

of i n t e s t i n a l ep i the l ium and r e t i c u l a r c e l l s of Ascar is

suum. He a lso s tudied the uptake of '^^C ATP a f t e r

1 4

incubat ing the uorms in Tyrode so lu t i on conta in ing C ATP

and found that r a d i o - a c t i v e l a b e l uas h igh ly concentrated

in i n t e s t i n a l e p i t h e l i a l c e l l s and sub-cut i cu lar t i s s u e s .

Ualker (1977) studied the r e l a t i o n s h i p between temp^^rature

change and mi tochondr ia l ATPase a c t i v i t y . ( d ) Carboxyl e s t e rases

Hart and Lee (1966) l o c a l i z e d the d i s t r i b u t i o n of

cho l i n e s t e r a s e a c t i v i t y and i n h i b i t i o n by anthe lmint ics

in var ious nematode pnrasiL<33. Ueznik (1 969) s tudied

the d i s t r i b u t i o n of o'-in-speci f i c oster-^ses in the body

u a l l of Ascar id ia g a l l i . Sanderson (1969) s tudied

cho l ines t r rasB a c t i v i t y in animal o^ ' r^s i t i c nematodes.

Erhnger d»d Crershon (1 970) studied cho l i nestn rase a c t i v i t y

in r e l a t i o n to the age of Turbntr ix a c e t i . Reznik and

Didenko (1 970) l o c a l i z e d the Cc rbmic estnrd3i?s in

c u t i c u l a r , sub-cut icu 1 ^ r nnd muscle, l ayers of the body

u a l l and al imentary t r a r t of ^-dult T r i chur i s niuris.

38

They found tha t n o n - c e l l u l a r c u t i c l e does not con ta in

c a r b o n i c e s t e r a s e s but the s u b - c u t i c u l a r l a y e r and muscle

l a y e r con ta in n o n - s p e c i f i c e s t e r a s e s and c h o l i n e s t e r a s e .

N o n - s p e c i f i c e s t e r a s e s uere found in c e l l s of a l imen ta r y

t r a c t .

Reznik (1971) found c o n s i d e r a b l e amount o f

c a r b o x y l i c e s t e r a s e in c u t i c l e , hypodermis , muscle and

i n t e s t i n a l e p i t h e l i u m of A s c a r i d i a q a l l i and A s c a r i s

l u m b r i c o i d e s . He fl97l) a l s o worked on ca rbon i c

e s t e r a s e s o f 3 p a r a s i t i c nematodes and found tha t

c u t i c l e of A s c a r i s suum and A s c a r i d i a g a l l i con ta ined

n o n - s p e c i f i c e s t p r a s e s 3nd tha t of T r i c h u r i s conta ined

c h o l i n e s t e r a s e . Cho l ines tn rase occurred in i n t e r c e l l u l a r

spaces o f gut e p i t h e l i u m of A s c a r i s suum and A s c a r i d i a

galli but not in Goithelium o f T r i c h u r i s . Ruitenberg

and Loonders lon t (1971) examinr-d l a r vae o f An i sak i s sp.

and found n o n - s p e c i f i c e s t . rase a c t i v i t y in c x c r o t o r y

organ and sugges ted tha t t h i s h i s t o c h e m i c a l t e s t may

be used f o r testing viability o f l a r va e and o f f e e t o f

a n t h e l m i n t i c s . They (1971) again studied the histochemical

nature o f e x c r e t o r y organ o f An i sak i s l a r vae and found

s t r o n g activity f o r n o n - s p e c i f i c e s t e r a s e s in l a t r r a l

p a r t o f the organ and moderatr activity in central a r ea .

Sanderson (I97l) l o c a l i B e d the distributiin o f

c h o l i n e s t e r a s e a c t i v i t y in some s p e c i e s o f S t r o n g y l o i d e a

and Oxyuro idea .

39

Tan and Zam ( i T T l ) found n o n - s p e c i f i c e s t e rases

in p c r i e n t r i c f l u i d of Ascar js suum. Gupta and Sood

(1974) domonstratod n o n - s p e c i f i c es t e rase act iv/ity in

the body wa l l of Hetorakis n u s i l l a -- nd discussed i t s

f u n c t i o n a l s i g n i f i c a n c e . rialyutina (1974) studied the

aco t ycho l ines t e rase system in nerve and muscle t i s sues

o f Ascar id i a p a l l i . Sanderson (1974) studied a c e t y l -

cho l i n e s t e r a s e in IMippostronqylus b r a s i l i e n s i s .

Goh and Davey (1176) studied nervous system of

Phocanema Hecipens using a c e t y l c h o l i n e method. They

found a c e t y l c h o l i n e s t e r a s e to be present ns smal l

d i s c r e t e granules in nerve r ing and 6 l ong i tud ina l nerve

cords . I j r ight and Aunn (1 976) de tec ted the cho l ines t e rase

a c t i v i t y in th^' nr-rvc r ing in i^anrgrel lus r e d i v i v u s ,

Cnenorhabdit is c l eqans , Prionchulus punctatus,

Aphelcnchus dvenau and TJitylonrhus d i p sac i . They found

that u l t r a son i c oretreatment of i n t a c t or cut nematodes

improved qro;^tly the st<' ining cons is tency at the nerve

r i n g . Yoat-3 and J g i l v i o (1976) s tudied the enzymic

p r o p e r t i e s nnd poss ib le p h y s i o l o g i c a l ro l e of a c e t y l -

cho l ines t e rase in Nippostrongylus b r a s i l i e n s i s and

Necator americanus.

Nuosu (197S) studied age r e l a t e d changes in

es te rase and aco ty1cho l ines te rnse a c t i v i t i e s of the

i n f e c t i v e larvae of Ancylostoma tubaeforma. Dang, Ning

40

and Uanq (1979) detbrmincd cho l inestL^rase a c t i v i t y in

aui3r 2000 i n t e c t Ancylostoma duodenalo and over 1000

Necntor amoricjnus rccovGred Fnm 2 05 pa t i en t s and

t r e a t e d u i th one nf thfj SGV' r a l ant ho In i n t i cs . Terenina

(1979) s tud ied the c h o l i n n s t i r a s e a c t i v i t y in female

g e n i t a l t r a c t s of 7 spec i es of nematodes in d i f f e r e n t

sub-o rde rs . The enzy^ie uas a c t i v e in areas of muscles

and ova.

( e ) A r y l sulphatase

This enzyme has m t so f a r been recorded in

nematodes. Anonq othf r he Im i nt iis Bog i tsch (1 966) s tudied

a ry I su lpho tase in Int^jrmediate l aye r of Hymenolepis

diminuta c y s t i c e r c o i d .

41

4. ^ \/itro cul t iv/at ion

Attempts by workers to c u l t i v a t e ^ ^/itro, the

m i c r o f i l a r i a e of d i f f e r e n t f i l a r i a l uorms, have met

LJith l i t t l e success . I n i t i a l l y the m i c r o f i l a r i a e were

c u l t i v a t e d in uhole b lood . In f a c t the c u l t i v a t i o n

in v i t r o of m i c r o f i l a r i a e of the f i l a r i a l nematodes

p a r a s i t i c in uarm blooded hosts uas f i r s t s u c c e s s f u l l y

c a r r i e d out by Hobson (1948 ) . This uork ujas rev iewed

by Ear l (1959) , uho c a r r i e d out a s e r i e s of iri v i t r o

exper iments on the maintenance o f the m i c r o f i l a r i a e of

D i r o f i l a r i a immi t i s , the dog heart worm, in a v a r i e t y

o f media at 37 C. The m i c r o f i l a r i a e surv i ved f o r

4 days in medium 199 alone but uihen supplemented u i th

10% i n a c t i v a t e d dog serum, the m i c r o f i l a r i a e surv i v ed

f o r 43 days at 37 C; they surv i v ed f o r 61 days when

serum supplement uas increased to 30%. He obta ined

s i m i l a r r e s u l t s uhen supplemented medium 199 with ox or

horse se ra . The r e s u l t s did not improve by using

kidney t i s sue cu l ture o v e r l a i d u i th medium 199 + 10%

i n a c t i v a t e d dog serum ( s u r v i v a l t ime 40 days o n l y ) .

Tay lo r (1 960a) c u l t i v a t e d ijn v i t r o , the

m i c r o f i l a r i a e of D. immit is in a v a r i e t y of media and

cu l tu r e s of mosquito uhole gut. She used medium 199

supplemented u i th horse serum, raw l i v e r or mosquito

e x t r a c t s , rod blood c e l l s -jr d i l u t e T y r o d e ' s s o l u t i o n

at 22 C, in an attempt to induce development of the

42

mosquito s t a g e s . The m i c n f i l a r i a e surv i ved f o r 14

days , no grouth or development occurred . She (1950a)

mainta ined ^ v i t r o , the m i c r o f i l a r i a e of Uuchereria

b a n c r o f t i and Loa loa f o r 10-14 days, in a v a r i e t y of

media conta in ing serum, blond c e l l s and some chemica l l y

d e f i n e d components. The m i c r o f i l a r i a e of U. b a n c r o f t i

exsheathed but no development occurred uhereas there

uaa an i n d i c a t i o n of the f i r s t s tage of development in

m i c r o f i l a r i a e of Loa l o a . In that she observed , the

G c e l l s 1 and 2, to have d i v i d e d .

In the f o l l o w i n g year 1961 Sawyer and Ue ins t e in

attempted to determine the importance o f var i ous

i n o r g a n i c ions f o r v i t r o s u r v i v a l of D . immit j s

m i c r o f i l a r i a e . He t e s t e d the i no r gan i c components of

E a r l e ' s , Gey ' s , Krebs-Ringer-phosphate and Locke 's

s o l u t i o n . In mod i f i ed Locke 's s o l u t i o n , the

m i c r o f i l a r i a e surv i ved f o r 35 h at 37 C and 55 h at

27 C. In Krebs-Ringer-phosphate u i th 0.25^^ g lucose

and 1.5;?^ sodium c a s e i n a t e , b u f f e r e d at nH 7 .2 , the

m i c n f i l a r i a e surv i ved f o r 5 days at 27 C but only

f o r 40 h at 37 C.

This uork was l a t e r extended by Sauyer and

Ue ins t e in (1 961, 1 9 6 3 a , b , c ) . They obta ined the

development of m i c r o f i l a r i a e to the l a t e f i r s t s tage

( e a r l y 'sausage s t a g e ' ) in culturt:s of hepar inated

b lood at 22 or 27 C. This development occurred as

43

e a r l y as 3 days i f cu l ture but no f u r t h e r development

occurred throughout the 21 day cu l ture pe r i od . They

c u l t i v a t e d in v i t r o , the m i c r o f i l a r i a e of Oipetalonema

in the some manner and obtainu^i 'sausage s t a g e ' a f t ^ r

12 days.

Sauycr and Ue ins te in (ig63b) incubated the

m i c r o f i l a r i a e of D. immit is at 22 or 27 C in human

blood or in r abb i t b lood or in Locke 's s o lu t i on

con ta in ing a suspension of dog blood c e l l s . Tho

development to 'sausage s t a g e ' uas obta ined in these

media. They a lso observed the s u r v i v a l o f m i c r o f i l a r i a e

f o r s e v e r a l weeks Liithnut development in dog plasma or

horse serum. In chemica l l y d e f i n ed cu l ture medium

(NCTC 109) they su r v i v ed only f o r 7 days and no

development occurred . Sawyer and Ue ins te in (1953c)

went on to f i nd the optimum concen t ra t i on of horse

serum which would promote the growth to the ' sausage '

form in NCTC 109. They obta ined the maximum y i e l d of

30^ at 27 C with 5-10f i n a c t i v a t e d serum in NCTC 1G9

and the s u r v i v a l t i n e increased to B days. A d e f i n i t e

inc rease in width a l so occurred in l e ss than 30 h of

i ncuba t i on . They found that gass ing the cu l tu r es

e v e r y 2-3 days with 5/'. CO2 in a i r was b e n e f i c i a l but

the medium was not changed during c u l t u r e . The

m i c r o f i l a r i a e did not deve lop at 37 C and the s u r v i v a l

time was reduced t i 4 days.

44

Weinste in (1963) app l i ed his technique f o r

c u l t i v a t i n g m i c r o f i l a r i a e of L i tomo3oides c a r i n i i .

Exshe^thnent uas induced by l y t i c enzymes o m i t t i n g

saponin which uas t i x i c . 'Sausage s t a g e ' uas obta ined

in 6 days in NCTC 109 u i th 20% i n a c t i v a t e d human serum

and a n t i b i o t i c s , supolemented u i th amino-ac ids , suaars

and organ ic ac ids of Grace 's medium. Here a lso he

found that the gass ing a i r u i th CQ2 uas b e n e f i c i a l .

Sauyer and Cheever (1962) r epo r t ed that

m i c r o f i l a r i a e found in Columbian marmoset (Oedipomidas)

su r v i v ed f o r 22 days in a suspension of marmoset

kidney c e l l s in medium 199 + c a l f serum. During t h i s

t ime m i c r o f i l a r i a e shoued an increase of 25% in l ength

but t h i s development uas supposed to be abnormal as

normal development to sausage s tage i s accompanied

by shor ten ing and f a t t e n i n g process .

Ue ins te in (1 963) c u l t i v a t e d ijn v i t r o the

m i c r o f i l a r i a e of Uucherer ia b a n c r p f t i in the same manner

as m i c r o f i l a r i a e of D i r o f i l a r i a immit is and obta ined

'sausaqo s t a g e ' in 4-7% of m i c r o f i l a r i a e . He added

a d d i t i o n a l amino-ac ids , sugars and o rgan i c ac ids to

supplement the medium, 40% of the m i c r o f i l a r i a e shoued

s i gns of development Jj]_ v i t r o . The time of Jevelopment

uas cons ide rab l y pro longed as compared to the time

r e q u i r e d in mrma l h i s t .

45

Plost s u c c e s s f u l c u l t i v a t i o n of m i c r o f i l a r i a e

has been achiev/ed by Uood and Su i t o r (1966) using

m i c r o f i l a r i a e of i'^acacanema formosana from the blood

of Taiwan monkey Hacaca c y c l o p j s . They used Aedes

a e g y p t i c e l l l i n e s o v e r l a i d u i th a medium con ta in ing

0.5^ haemolymph from Phi losamia cynth ia + 10^ f o e t a l

bov ine serum in Grace ' s i n s ec t c e l l cu l ture medium GHA,

P l i c r o f i l a r i a e uere separated from hepar inated blood

by t reatment with saponin b e f o r e being cu l tu r ed . The

development to t h i r d s tage took p lace at 22 C but not

at 28 C. [Medium uas not changed throughout the 33 day

cu l tu r e pe r i od . Liithin 5 days the l a rvae had reached

'sausage s t a g e ' and the movement uas maximum. Between

6-9 days la rvae grew r a o i d l y in l ength and narrowed

s l i g h t l y and in 14-19 days the l a rvae reached the

i n f e c t i v e s t a g e .

'Jc instoin (1970) r epor t ed the deve l ipmont of

Dinetalonema reconditum m i c r o f i l a r i a e to 'sausage' f )rm " • ,

us ing uholc blood and blood f r a c t i o n s from ;iog and

other mammals.

A ik i (1971) r epor t ed a c t i v e exsheathment of

Uucherer ia b a n c r o f t i and Brugia pahangi m i c r o f i l a r i a e

in th i ck blood f i l m s , kopt in moist chambers and on

agar p l a t e s at d i f f e r e n t t emperatures , s a l i n i t i e s and

oH. At 15-20 C, more than 50% m i c r o f i l a r i a e of

46

U. banc r o f t i exshGathed in 4 h, 80% in h and 90% in

12 h. On agar platGs 90% nf U. b a n c r o f t i m i c r o f i l a r i a e

cxsheathed u i th in 8 h and 98.2^ of B. pahangi did so in

1 h. The rate dec l ines at higher temperature. Ib'hen

agar p la te cu l tures uere exposed at 20 C f o l l o u i n q 8 h

s to rage at 5 C the exsheathment ra te uiaa a c c e l e r a t e d .

Lack of sa l ine or concentra t ions ouer 2% k i l l e d the

m i c r o f i l a r i a e . The best concentrat ion was D.9% and

optimum pH uas 5.8 - 6.8.

Cupp (1972) uorking in a Uorkshop on Dev/elopment

of F i l a r i a c in Hosquitoos, U.S.-Japan Co-oporativ/e

Medical Science Program, Un i ve r s i t y of C a l i f o r n i a ,

Los Ange les , has repor ted development to l a t e L

s tage in D, immit is and D. corynodes in mosquito c e l l

l i n e s uhich appeared enhanced by i nsec t haemolymph.

luamoto (1972) made ex t ens i v e j j i v i t r o

Gxperiments with D i r o f i l a r i a immit is and Uuchercria

b a n c r o f t i . He found that 50% of D. immitis micr i f i 1 a r i r u

surv ived f i r 216 h at 5 C, 61 h at 21 C and 9 h at

37 C in s a l i n e . He fiiund that 100% m o r t a l i t y iccurred

at 426, 352 and 48 h of c u l t i v a t i i n , r e s p e c t i v e l y . At

21 C 50% surv ived f i r 74 h in d i s t i l l e d uator , 61 h

in s a l i n e and 216 h in rabb i t plasma and 254 h in

plasma of un in fec ted dogs. He found that s u r v i v a l time

o f U. b a n c r o f t i m i c r o f i l a r i a uas shor ter than D. immitis

47

m i c r o f i l a r i a at a l l temperatures and in a l l c o n d i t i o n s .

In t ravenous t r a n s f u s i o n of D. immit is i n t o dogs shouerl

m i c r o f i l a r i a o t b o present in p e r i p h e r a l c i r c u l a t i o n

f i r at l e a s t 50 days. Houever in r a b b i t s , m i c r o f i l a r i a e

d isappeared a f t e r 21 days. U. b a n c r o f t i m i c r o f i l a r i a e

cou ld not be demonstrated in p e r i p h e r a l c i r c u l a t i o n of

r a b b i t s or dogs, but on autopsy immodiatoly a f t e r

giv/ing i n f e c t i o n moderate number of m i c r o f i l a r i a e were

found in lungs, l i v o r and k idneys .

K le in and Bradley (1974) c u l t i v a t e d m i c r o f i l a r i a e

of D. immit is in NCTC 109 f o r 2-7 days and f o r 6-17 days

in Grace 's medium supplemonted with 20% heat i n a c t i v a t e d

horse serum. The la rvae showed stomal and anal p l a t e

f a r beyond those seen in L s t a g e , yet no e c d y s i s

occur red .

riuaiko and f^kufya ( i g 7 4 ) used s e v e r a l media f o r

c u l t u r i n g in v i t r o , the m i c r o f i l a r i a e o f Onchocnrca

V o l vu lus and G. qut turosa . They .ibserved the

m i c r o f i l a r i a e to su rv i v e f o r lonqnst pe r i od if time

(8-10 and 5-6 days r e s p e c t i v e l y ) in d i s t i l l e d uat r

c o n t a i n i n g 10% c a t t l e serum and 3% g lucose . D i lu t e

T y r o d e ' s s o lu t i on 1:1 v/v u i th d i s t i l l e d uatcr

c on ta in ing 1 0/m c a t t l e serum was found to be e q u a l l y

s u i t a b l e . Devaney and Houel ls (1978) s tud ied the

development in v i t n of a r t i f i c i a l l y exsheathed

m i c r o f i l a r i a e of Brugia pahangi in c o l l l i n e s de r i v ed

48

from Aedes s c u t e l l a r j s , maiayens is , A. acgyp t i and

Amohe l e s s t ephans j . Thoy f jund thot the 'sausage

s t a g e ' icrua dcvc l ipod in these exper iments , extrudes

c e l l s frnm anal pir .- . Thoy ( i g 7 9 ) s tud ied the

devc l ipment i f J r t i f i c i ' l l y exshe-^thed m i c r o f i l a r i a e

of Brugia pahangi and malayi in rrusquiti c e l l l i nes

of 3 spec i es Df Aedos and Anopheles s tephens j at 28 C. ody

Deuelopment :5f m i c r a f i l a r i ae of B, roalayi c o u l ^ b e

s tud ied in Ae des malayensis due t i the lack i f a v a i l a b l e

number nf p a r a s i t e s . Na development occurred in the

absence of mosquito c e l l s . The best r e s u l t s uere

obta ined with A. malaye nsis f o r both B. malayi and

B. pahangi. Up tD 13% ^f larvae reached 'sausage s t a g e '

in 8 days f i r B. pahangi and up to 30^ in 6 days f o r

B. malay i . They (1979) c a r r i ed out amthe r experiment

using cu l ture system fo r maintenance and development

of m i c r o f i l a r i a e if Brugia pahangi and D i r o f i l a r i a

i n r i t i s . In medium NCTC 135 supplemented with 20%

heat i n a c t i v a t e d FB5, 15-20^' i f micr i f i l a r i a e deve loped

t i l a t e f i r s t stage l a r vae . I n c lus i on if insuct c o l l s

d id not impr ive the culturt^s.

Dcvanuy and H^uells ( l g 7 9 ) induced the Gxshe athment

^ ^ ^ Pahangi m i c r o f i l a r i a e in v i t r o . They i s i l a t e d

the m i c r o f i l a r i a e fr-^m C ' t b l -nd and incubat fd f - r 1 h

49

in 20 mH CaCl2 in phosphatG-froe Hank's BSS. A l t e r n a t i v e l y

they UBTG incubstRd f i r 30 minutes in s o l u t i o n s of

endopopt idase (5 .8 uni ts/ml ) or papaya e x t r a c t

protG.^se (3 .0 uni ts/ml ) in c a l c i u m - f r e e BSS. The same

techniques are e f f e c t i v e for m i c r o f i l a r i a e of

Uucherer ia b a n c r o f t i and L i tomosoides c a r i n i i .

50

STATEMENT OF PROBLEM

I t can be seen from the f o r e go inq account of

h i s t o r i c a l r e v i eu that our knowledge on var ious aspects

of f i l a r i a l worms i s qu i t e inadequate and whatever work

has been done i s meagre as w e l l as s c a t t e r e d . The

morphology of d i f f e r e n t spec i es of f i l a r i a l uorns has

been s tudied by a number of workers only by l i g h t

microscopy . The f i n e c u t i c u l a r s t ruc tu r e s by s t e reoscan

e l e c t r o n microscopy, have only been s tud ied in a feu

s p e d e s .

The present work rjyals with a d e t a i l e d

r e r i e s c r in t i on of Chandlere11a haukingi C h a t t e r j i , Sen

and Bhattacharya, 1965; with a d d i t i o n a l d e t a i l s

e s p e c i a l l y those which have been 'hne by s t e reoscan

e l e c t r o n microscopy.

The present knowledqe on the s t ruc tu re of

m i c r o f i l a r i a e i s a l so incomple te . The nuclear land

merks l i k e f i r s t c e l l of nuslear column, nerve r i n g ,

e x c r e t o r y c e l l , H - c e l l , h - c e l l s , anal c e l l , l a s t c e l l

o f nuclear column and orqansof Schwanzqebilde are

poo r l y descr ibed in known poec ies . The c e p h a l i c hook

and sp ines , the o r a l r i n g , the pharynqeal thread and

Innenkoroer are known only in a few s p e c i e s .

51

In the present i n v e s t i g a t i o n , the nuclear

s t ruc tu r e s of m i c r o f i l a r i a of C, hauking i , have been

r e d e s c r i b e d , because in the o r i g i n a l d e s c r i p t i o n , by

C h a t t e r j e e , Sen and Bhattacharya (1965 ) , the f i g u r e

does not shou a c l e a r p i c tu r e o f the nuclear landmarks

which are of g rea t importance in i d e n t i f y i n g or

c l a s s i f y i n g the genera of f i l a r i a l worms.

Sometimes the nuclear s t ruc tu r e s are not

s u f f i c i e n t enough f o r i d e n t i f i c a t i o n of genera, the

c e p h a l i c s t ruc tu r e s which are spec i f i c f o r d i f f e r e nt

genera , have a l so been s tud ied in m i c r o f i l a r i a of

C. hawkingi .

The r e l a t i v e s i z e and p o s i t i o n of Innenkorper

i s a l so of g rea t va lue in the c l a s s i f i c a t i o n of f i l a r i a l

worms and hence the o r a l r i n g , the pharyngeal thread

and the Innenkorper have a lso been s tud ied in long as

w e l l as short forms of m i c r o f i l a r i a e of C, hawkingi .

I t has a lso been proved that the long and the

shor t forms of m i c r o f i l a r i a e belong to C. hawkingi

o n l y , by g i v i n g r a t i o s of body length and s i z e o f

Innenkorper , r e l a t i v e d is tance of Innenkorper , from

a n t e r i o r and p o s t e r i o r ends of the body.

The work on d e t a i l e d h i s t o l o g y of d i f f e r e n t

systems of f i l a r i a l worms i s a l so l i m i t e d to only two

s p e c i e s , Brugia pahangi and S e t a r i a c e r v i . The r e s t

52

of the uork i s s c a t t e r e d and unsystemat ic . In the

f o l l o u i n g pages an attempt has been made to study the

d e t a i l e d h i s t o l o g y of the body w a l l , the musculature,

the d i g e s t i v e and r ep roduc t i v e systems of C. hauking i ,

in v i eu that c e r t a i n h i s t o l o g i c a l cha rac t e r s , l i k e

musculature , the nuclear constancy of oesophagus, the

c e l l s of the i n t e s t i n e prov ide important c r i t e r i a f o r

c l a s s i f y i n g nematodes.

The uork on enzyme l o c a l i z a t i o n i s s c a t t e r e d and

inadequate . As the enzymes are ve ry s p e c i f i c in t h e i r

d i s t r i b u t i o n in d i f f e r e n t genera and s p e c i e s , t h e i r

l o c a l i z a t i o n p rov ides a good taxonomic t o o l f o r

s e p a r a t i n g d i f f e r e n t f i l a r i a e . I n v e s t i g a t o r s showed

s t r i k i n g l y d i f f e r e n t pa t t e rns of phosphatases, comrionly

ac id and a l ka l i n e phosphatases in m i c r o f i l a r i a e of

d i f f e r e n t genera and s p e c i e s . In view of t h i s , the


t r i phospha tas e , ca rboxy l e s t e rase and a r y l su lpha tase ,

have been s tud ied in m i c r o f i l a r i a e of £. hauking i .

Only four of these , the ac id phosphatase, a l k a l i n e

phosphatase^ adenosine t r iphosphatase and ca rboxy l

e s t e r a s e have been s tud ied in adu l t .

The Uork on in v i t r o c u l t i v a t i o n has been done

on a feu spec i es only and yet not very s u c c e s s f u l .

In the present uork the in v i t r o c u l t i v a t i o n of

53

m i c r o f i l a r i a e of C. haukingi has besn done as these

s tud i e s are of g rea t importance f o r

( a ) s tudying the deve lopmenta l s tages outs ide

the body of the v/ector,

(b ) s tudy ing the e f f e c t of var ious chemicals

and drugs on adult as u e l l as l a r v a l

s tages of the p a r a s i t e , and

( c ) i t may a l so help in the c o l l e c t i o n of ES

products uhich may serve as f u n c t i o n a l

ant igens aga inst f i l a r i a s i s , as somatic

ant igens have f a i l e d to immunise the

i n d i v i d u a l s .

54

(MATERIALS AND PIETHODS

1. Olorphology


( i ) Adult

The adult uorms uere c o l l e c t e d from the hear t

in luke uarm normal s a l i n e (0.8?S NaCl ) e i t h e r by

d issect ing an anaes the t i s ed crou or a d e c a p i t a t e d l i v e

c rou . The heart uas taken out in normal s a l i n e ,

d i s s e c t e d and examined f o r the uorms. The body c a v i t y ,

uhich ge t s f u l l o f b l o od , was a l s o thorough ly uashed

u i t h normal s a l i n e and the con ten ts uere examined f o r

the uorms, as most o f the males escape uhen the heart

i s cut and taken out . The uorms uere uashed thorough ly

in normal s a l i n e and f i x e d in hot 70/S a l c o h o l .

For s tudy ing gross morphology, the uorms uere

c l e a r e d in g l y c e r i n e a l c o h o l , l a c t o p h e n o l or c r e o s o t e .

They uere then p laced on a s l i d e under a c o v e r - g l a s s and

manipulated by r o l l i n g them in c l e a r i n g agent .

D i f f e r e n t s t r u c t u r e s uere s tud i ed under the m ic roscope .

The c e p h a l i c end uas d e c a p i t a t e d and mounted in

e n - f a c e \yieu in g l y c e r i n e - j e l l y f o r s tudy ing c e p h a l i c

p a p i l l a e and amphids.

55

( i i ) n i c r o f i l a r i a

Nuclear s t ruc tu res

For s tudy ing the nuclear s t ruc tu r e s of mic ro -

f i l a r i a e , the smears uere made by tak ing the blood from

uing uein at n i gh t . The smears uere dehaemoglob in ised,

d r i e d in a i r and d i r e c t l y s ta ined u i th Leishman's s t a in

or they uere f i x e d e i t h e r in a c e t i c a c i d - f o r m a l i n -

a l c o h o l (AFA) , methyl a l c oho l or ace tone . The s t a ins

used uere e i t h e r Giemsa's s t a i n , haematoxy l i n - eos in ,

H a l l o r y ' s t r i p l e or H a l l o r y ' s phospho- tungs t i c a c i d -

haematoxy l in .

Cepha l i c s t r u c t u r e s , pharyngeal thread and Innenko'rper

For s tudy ing the c epha l i c s t r u c t u r e s , pharyngea l

thread and Innenko'rper, the smears uere made u i th the

b lood taken from uing ve in Pt n i gh t , from hepar ipated

b lood from heart or from m i c r o f i l a r i a l concen t ra t i ons

suspended in hepar in i sed plasma.

H i c r o f i l a r i a l concen t ra t i ons uere prepared by

l y s i n g RBCs u i th saponin. 5 ml of 0.5% s o l u t i o n of

saponin in normal s a l i n e uas added to 0.5 ml of i n f e c t e d

b lood and kept at 32 C f o r 10 minutes. The contents

uere c e n t r i f u g e d at 15oO rpm f o r 8 minutes, the

supernatant uas taken out and 8 ml of R i n g e r ' s s o l u t i o n

uas added to the sediment , thoroughly mixed and

c e n t r i f u g e d at 1500 rpm f o r 10 minutes. Three such

56

washings were g iven by c e n t r i f u g i n g and d i s ca rd ing the

sunernatant . Then the sediment was suspended in

hepa r in i z ed plasma and smears were made, d r i ed and f i x e d

usua l l y in a c e t i c a c i d - f o r m a l i n a l c o h o l (AFA). Other

f i x a t i v e s used were e t h y l a l c oho l 90% (warm), methanol :

ch lo ro f o rm (2 :1 m/m), Zenker ' s f l u i d or Bouin Dubosq.

The f i x a t i v e was washed thoroughly in running tap water

f o r 4-5 h. The smears were then sub j e c t ed to l i p i d

e x t r a c t i o n by keeping the s l i d e s o ve rn i gh t in methanol :

ch lo ro f o rm (2:1 v / v ) at 50 C and then in absolute

a l c o h o l f o r s e v e r a l days.

A f t e r l i p i d e x t r a c t i o n the smears were sub j e c t ed

e i t h e r to o x i da t i on or su lpha t i on . The o x i d a t i o n was

done with a c i d i f i e d permanganate (0.3% KHnO^ with 0.2 ml

conc. H^SO^ per 100 ml) or p e r i o d i c ac id . The l a t t e r

d id not g i ve very s a t i s f a c t o r y r e s u l t s . The su lphat ion

was done by dehydrat ing the smears in graded e t h a n o l s ,

then immersing them in g l a c i a l a c e t i c ac id f o r 1 minute,

f o l l o w e d by keeping in a mixture of conc. H2S0^ and

g l a c i a l a c e t i c ac id (1:1 v/v ) f o r 20 minutes at room

temperature . The smears were then washed s u c c e s s i v e l y

in g l a c i a l a c e t i c a c i d , tap water and d i s t i l l e d wa te r .

The ox i da t i on was done so as t o in t roduce the

a c i d i c group i n t o the s t ruc tu r e s in m i c r o f i l a r i a e .

A f t e r o x i d a t i o n the smears were s t a ined with bas i c dyes

at low pH. Aldehyde fuchs in 0.5% in 70% e thano l with

57

1.0 ml conc. HCl/10n ml; bas ic fuchs in 0.^% in 0.05 N HCl;

c r y s t a l v i o l e t 0.1% in 0.05 N HCl; neu t ra l red 0.5^ in

70% e thano l u i th 1 ml conc. HC1/10Q ml; a l c i an blue

in 3% a c e t i c ac id and b r i l l i a n t c r e s y l blue Q,1/S in

0.05 N HCl.

The study o f the long and short forms of mic ro -

f i l a r i a e uas done u i th these p r epara t i ons on l y .

Measurements were taken u i th the help of an ocular

micrometer and are in m i l l i m e t e r s unless s t a t ed o t h e r u i s e .

The drauings uere made u i th the help of a Camera luc ida

at proper m a g n i f i c a t i o n . Flany d e t a i l s uere added in

the f i n i s h e d drauings uhich uere inked by Ind ia ink ,

l a b e l l e d and photographed.

58

( b ) Stereoscan study

( i ) Adult

The adult uorms uere taken out from the heart of

anaes the t i s ed crous. They uete kept and thoroughly

washed in normal s a l i n e ( 0 . 8 ^ ) . The uorms uere then

kept in r e f r i g e r a t o r at 4 C so that they become s l u g g i s h .

A f t e r about 1 h the worms uere f i x e d in g lu ta ra ldehyde

in 0.1 n phosphate b u f f e r and l e f t o ve rn i gh t at 4 C in

a r e f r i g e r a t o r . Next morning the f i x a t i v e uas washed

u i th 0.1 n phosphate b u f f e r f o r 10-12 h u i th at l e a s t

5 changes. The worms were then doubly f i x e d with

C a u l f i e l d ' s osmium t e t r a - o x i d e s o l u t i o n f o r 1 h at

4 C. T h e r e a f t e r they uere washed thoroughly with

d i s t i l l e d water and dehydrated in graded a l c o h o l s , i » e . ,

through 30%, 5fJ%, 70%, 80%, qn|% and 95% f o r 10 minutes

each and then tw ice in dry a l c o h o l f o r 30 minutes each.

A f t e r dehydrat ion was complete , the worms were passed

through graded amyl ace ta te s o l u t i o n , 30%, 50%, 70%,

80%, 90%, prepared in abso lute a l c o h o l , f o r 10 minutes

each, f o l l o w e d by two changes in absolute amyl ace ta te

f o r 30 minutes each. Amyl ace ta t e was used as i t i s

m i s c i b l e with both a l c o h o l and l i q u i d CO2, hence i t i s

necessary to in t roduce i t in the t i s sue be f o r e c r i t i c a l

po in t dry ing with l i q u i d C02* The worms uere then

c r i t i c a l l y d r i ed in Balzc^rs Union Type H 9202 C r i t i c a l

po in t D r i e r . The des i r ed par ts were mounted on stubs

59

wi th s i l v e r dag and d r i e d . They uere l a t e r coated

wi th a go ld -pa l l ad ium a l l o y in a Sput ter coa te r

( Po l a ron SEH Coat ing Unit E 5000) . The specimens ucre

observed under Cambridge Stereoscan 180 Scanning

E l e c t r o n r i i c roscope . Photographs u/ere taken an Indu

120 r o l l f i l m of a speed of 125 ASA.


The m i c r o f i l a r i a e were f i r s t separa ted from the

uholG bloot i . For t h i s 50 mg phytohaemagg lut in in ( D i f c o )

uas d i s s o l v e d in 5 ml o f d i s t i l l e d u a t e r . 1 ml pf t h i s

s o l u t i o n uas mixed with 9 ml o f d i s t i l l e d u a t e r . Th is

was used as the s tock s o l u t i o n o f phytohaemagg lut in in .

For s e p a r a t i n g m i c r o f i l a r i a e 0.2 ml of t h i s

s o l u t i o n uas added to 0«5 ml of b lood and the tube was

g e n t l y r o t a t e d f o r 2 minutes f o r mixing p r o p e r l y the

phytohaemagg lut in in wi th the b lood . To t h i s 5 ml of

R i n g e r ' s s o l u t i o n uas addod and c e n t r i f u g e d at IDOO rpm

f o r 3 minutes. The conglomurated RBCs, uhich become

heav i e r u i th phy tohaemagg lu t in in , s e t t l e doun in the

bottom and the c l e a r supernatant , having m i c r o f i l a r i a e ,

was c a r e f u l l y t r a n s f e r r e d to a c e n t r i f u g e tube. This

supernatant was c e n t r i f u g e d at 1500 rpm f o r 10 minutes.

From t h i s the supernatant uas d i scarded anci the sediment

uas washed t h r i c e wi th R i n g e r ' s s o l u t i o n by c e n t r i f u g i n g

and d i s c a r d i n g the supernatant . Thus a l l the t r a c e s o f

phytohaemagg lut in in were removed.

60

The separated m i c r o f i l a r i a e uere f i x e d in 4%

g lu ta ra ldehyde s o l u t i o n in 0.1 n phosphate b u f f e r f o r

24 h at 4 C in r e f r i g e r a t o r . They uere washed t h r i c e

in 0.1 n phosphate b u f f e r by c o n t r i f u g i n g and d i s ca rd ing

the supernatant . They uere then doubly f i x e d in

C a u l f i e l d ' s 1% osmium t e t r a - o x i d e f o r 1 h at 4 C. A f t e r

osmioat ion the m i c r o f i l a r i a e uere uashed thoroughly in

d i s t i l l e d uater and dehydrated in graded e thano l a , i . e . ,

through 50^, 80%, 9^% mi f o r 10 minutes

each and then tu i c e in dry a l c o h o l f o r 30 minutes each,

e v e r y t ime by c e n t r i f u g i n g and d i s ca rd ing the supernatant .

A f t e r dehydrat ion i s complete , the m i c r o f i l a r i a e uere

t r a n s f e r r e d in 1:1 s o l u t i o n o f abso lute a l c oho l and

propy lene ox i d e . They uero resuspended in t h i s l i q u i d

by c e n t r i f u g i n g and d i s ca rd ing the supernatant . A drop

of t h i s l i q u i d having m i c r o f i l a r i a e uas put on a cov/er-

g l a s s , d r i ed in a i r and mounted on specimen stub with

s i l v e r dag. These uere then coated wi th go ld -pa l lad ium

a l l o y in a Sputter coa te r (Po laron SEN coa t ing Unit E 5000) ,

The obse r va t i ons uere made under Cambridge Stercoscan

180 Scanning e l e c t r o n microscope , photographs uere taken

on Indu 120 r o l l f i l m of a speed of 125 ASA.

61

The adult uorms uere c o l l e c t e d in luke uarm

normal s a l i n e (0 ,8^ NaCl) from heart oP an anaes the t i s ed

or decap i t a t ed crouj. They were s t r a i gh t ened in hot

(60-65 C) normal s a l i n e and f i x e d in aqueous Bouin 's or

a l c o h o l i c Bouin 's Dubosq f i x a t i v e f o r a pe r i od of 14-18 h,

A f t e r f i x a t i o n , they were t r a n s f e r r e d to a l c o h o l ,

uhich uas a l so used f o r s t o rage . When des i r ed they were

dehydrated , c l e a r e d and embedded in T i ssu mat (55 C) .

The s e r i a l s e c t i o n s , cut with the help o f a blade

attachment (AO), were f l a t t e n e d on albuminised s l i d e s

and rou t ine techniques uere used f o r s t a i n i n g . DPX uas

used f o r f i n a l l y mounting the s e c t i o n s .

The s ta ins used uere l a y e r ' s haemalum, Harr is

haematoxy l in , Heidenhairts haematoxyl in u i th which i r on

alum 3/? uas used as mordant and 1.5^ f o r d i f f e r e n t i a t i o n .

Eosin uas used f o r c o u n t e r s t a i n i n g . Other s t a in s used

are U o i g e r t ' s I ron haGmatoxylin, counter s t a i n used

be ing Van C i eson ' s p i c r o - f uchsin, l ^ a l l o r y ' s phospho-

t u n g s t i c ac id haematoxy l in . P i c r o f u c h s i n method of

Ka t t ine (1962) uas a lso t r i e d to s t a i n the nuc l e i and

n u c l e o l i . In t h i s method the mordant used c ons i s t s o f

phosphotungst ic and phosphomolybdic ac id (1 ,25^ s o l u t i o n )

i n 1:1 r a t i o (u/v ) .

62

For g e t t i n g ba t t e r d i f f e r e n t i a t i o n , i r on ton ing

uos done. The s e c t i ons were t r e a t e d u i th 1.5%

a f t e r s t a i n i n g u i th hasmatoxyl in and r i n s i n g in d i s t i l l e d

wa t e r . This makes the nuc l e i b lack .

M a l l o r y ' s t r i p l e s t a in was a l so t r i e d . The best

r e s u l t s uere obta ined u i th naye r ' s haemalum which u/as

qu i t e quick a l s o . P i c r o f u c h s i n method a lso gave good

r e s u l t s .

The s e c t i ons were examined under microscope.

Camera luc ida drawings of the s e c t i o n s pass ing through

d i f f e r e n t important r eg i ons were made. The drawings

were inked with Ind ia ink , l a b e l l e d and photographed.

63

3. H is tochemis t ry

( i ) Adult

The adult worms uere taken out in co ld normal

s a l i n e . Thoy uere washed t h r i c e u i th co ld s a l i n e so as

t o remove the deb r i s present on th&ir body. The f i xa t i o r^

uas done in co ld b u f f e r e d formaldehyde f o r 18 to 24 h

at 0-4 C. The worms uere t r a n s f e r r e d to gum sucrose

and kept f o r 12-24 h at 0-4 C and then washed b r i e f l y

in co ld d i s t i l l e d water . T h e r e a f t e r a g e l a t i n block was

made. The embedding was done in g e l a t i n f o r 1 h at

37 C. The block was made in a smal l me ta l i c cup. I t

was then coo led in a r e f r i g e r a t o r , when g e l a t i n had

s o l i d i f i e d comp l e t e l y , the block was hardened in 4%

f o rma l i n f o r 1 h. A f t e r hardening, the block was

trimmed t o the des i r ed s i z e . I t uas kept on a holder

a long with a drop of water under i t and a l lowed to

f roGzc with l i q u i d CQ2 on a carbon d i ox ide bench f r e e z e r .

Uhen the block has p rope r l y f r o z en i t was f i x e d on a

f r e e z e bar in c r y o s t a t . The micrometer was se t at 10 yu

t h i c k n e s s . The s e c t i o n s were cut by a S lec Type HS

Cryos ta t (Cambridge Hocking Microtome) and taken e i t h e r or

d i r e c t l y on c lean s l i d e s ( p r e c h i l i e d ) / i n gum sucrose

s o l u t i o n . For p i ck ing up the s e c t i o n s d i r e c t l y on the

s l i d e , a cover s l i p suc t ion p ick-up was employed.

Clean cover s l i p s or s l i d e s were p icked up by

squeez ing the t e a t and e x p e l l i n g the a i r from c a p i l l a r y

tube . The sucker d i s c was then app l i ed to cov/er s l i p

or s l i d e and tho t e a t uas r e l e a s e d a f t e r p i ck ing up the

s e c t i o n s by p ress ing the s l i d e f i r m l y and s t e a d i l y on

the sur face of the k n i f e .

These s l i d e s unro dr i^d at room temperature f o r

2 h. A f t e r the s e c t i o n s had d r i ed and adhered p r ope r l y

t o the sur face of the s l i d e they were incubated in the

d e s i r e d s u b s t r a t e .


Tuo methods uera used.

( 1 ) r^odif iud l ead n i t r a t e method

(2 ) Coupling azo-dye method

( 1 ) Mod i f i ed load n i t r a t e method

Sec t ions uure incubated f o r one ha l f to tuo hours

in the f o l l o w i n g s o l u t i o n :

2 v o l s 2% sodium- 5 - g l y c e r o p h o s p h a t e

1 v o l 0.1 .^ -ace t f t e b u f f e r (pH 5 -6 )

1 v o l 2.% l ead ace ta t e

0.3 v o l 1-5% P1gCl2

They were then r insed in d i s t i l l e d uatur and

deve loped in ammoniacal s i l v e r n i t r a t e s o l u t i o n f o r

30 minutes (28?^ ammonia water was added drop by drop to

5% aqueous AgNO^ u n t i l l the p r e c i p i t a t e j u s t d i s s o l v e s )

and r insed in 5% sodium th i osu lpha te f o r 5 minutes and

mounted in g l y c e r i n e j e l l y .

65

) Coupling azo^dye method using hexazo t i z ed p a r a r o s a n i l i n coupler

This method i s qu i t e quick and s e n s i t i v e . The

s e c t i o n s were incubated in a mixture conta in ing 10 mg r\-

naphthyl phosphate in 10 ml of ace ta te b u f f e r . To uhich

750 mg of p o l y v i n y l p y r r o l i d on K60 uas added and s t i r r e d

u e l l t i l l i t became homogeneous. To t h i s 1.0 ml of a

mixture conta in ing 0.5 ml NaN02 + 0.5 ml HPR uas

added and s t i r r e d . The s o l u t i o n was a l lowed to stand

b e f o r e being added to the incubat ion mixture . The pH

uas ad justed t o 5. The incubat ion mixture having t h i s

s o l u t i o n uas f i l t e r e d d i r e c t l y on to the s e c t i o n s and

incubated at 37 C f o r 30 minutes. The s e c t i ons uere

uashed in running water f o r 2 minutes and mounted in

g l y c e r i n e j e l l y .

( b ) A lka l ine phosphatase

Two methods f o r a l k a l i n e phosphatase uere t r i e d .

( 1 ) Gomori 's method

(2 ) Mod i f i ed coup l ing azo-dye method

( 1 ) Gomori 's method

The s e c t i ons uere incubated f o r one ha l f to tuo

hours in the f o l l o u i n g subst ra te c o n t a i n i n g :

10 ml of 3 0 sodium yS-glycerophosphate

10 ml of 2% Sodium d i - e t h y l ba rb i tu ra t e

5 ml d i s t i l l e d uater

20 ml magnesium sulphate

66

They were then r insed in water and t r e a t e d with

2% coba l t s o l u t i o n f o r 5 minutes. They uere washed in

uatsr and t r e a t e d wi th y e l i o u amirionium sulphide ( d i l u t e )

f o r 15 minutes, counte rs ta ined in aqueous eos in f o r

5 minutes and uashed in running water and mounted in


(2 ) Mod i f i ed coup l ing azo-dye method

The s e c t i ons were incubated in a mixture made by

d i s s o l v i n g 15 mg (10-20 range ) sodium o(-naphthyl phosphate

i n 20 ml of 0.1 PI stock ' t r i s ' b u f f e r (pH 10 ) . To t h i s

20 mg of the s t ab l e d i a z o t a t e o f 5 - c h l o r o - O - t o l u i d i n e

( s a l t 9 t ab l e 55 Pearse (1968) Diazonium S a l t s in

Enzyme H i s t o chem i s t r y ) was added and s t i r r e d w e l l . I t

was f i l t e r e d d i r e c t l y on to the s e c t i o n s in a s u f f i c i e n t

quan t i t y to cover them at 20 C (range 17-22 C) in a

BOD incubator f o r 15-60 minutes. A f t e r that they were

washed in running water f o r 1-3 minutes and counter

s t a in ed in f l aye r ' s haemalum f o r 1-2 minutes. A f t e r t h i s

the s e c t i ons were washed in running tap water f o r

30-60 minutes. The s ta ined s e c t i ons were mounted in


( c ) Adenosine t r iphosphatase

The s e c t i ons were sub j ec t ed to incubat ion f o r 3 h

in abso lu t e l y f r e sh medium c o n t a i n i n g :

67

0.1 n-sodium ba rb i tu ra t e (2.062 g/100 ml) 20 ml

0.1 Fl-CaCl^ (1.998 q/lOO ml) 10 ml

D i s t i l l e d water 30 ml

Adenosine t r iphosphate (disodium s a l t ) 152 mg

( F i n a l concen t ra t i on 0.005 Pi)

pH UBS ad justed to 9.4 u i th 0.1 PI >JaOH the

mixture uas brought to 100 ml u i th d i s t i l l e d uater

and f i l t e r e d , i f t u r b i d .

A f t e r the incubat ion uas complete the s e c t i ons

uere washed in 3 changes of 1"'' CaCl^, kept in 7.% CoCl^

f o r 3 minutes, uashed b r i e f l y in d i s t i l l e d water and

deye loped in d i l u t e y e l l o u ammonium su lph ide , uashed

tho rough l y , dehydrated, c l ea r ed and mounted in UPX.

( d ) Carboxy lesterasG

Hnly one method i . e . HPR method was used to

demonstrate c a r b o x y I p s t e f a s e in adul t worms. In t h i s

the s e c t i ons were sub j ec t ed to incubat ion in thr

f o l l o w i n q subst ra te at pH 6.5.

7 .5 ml of f].2 f'T phosohate b u f f e r stock s o l u t i o n

2.5 ml d i s t i l l e d wati^r

0.25 ml subst ra te s o l u t i o n (l/J o^-naphthyl

ace ta te in ace tone )

The mixture was gen t l y shaken and 0.8 ml of

HPH was added. This mixture was d i r e c t l y f i l t e r e d on

to the sec t i ons and incubat ion was c a r r i e d out f o r

30 minutes at 30 C. The s e c t i ons were washed b r i e f l y

in wate r , dehydrated in graded e thano ls and mounted in DPX

BB

( i i ) n i c r o f l l a r i a

Smears uicre made u i th the n ight blood from uing

v e in of heav/ily i n f c c t e d crow. They uere d r i ed in a i r

f o r 10-15 minutes, dehaemoglobin ized in co ld R i n g e r ' s

s o l u t i o n f o r 3-5 minutes and then f i x e d in b u f f e r e d

sucrose f o rma l in (pH 7 . 2 ) f o r 10-15 minutes at Q-4 C,

r insed in gum-sucrose s o l u t i o n and s t o r ed in the same

f o r o ve rn i gh t or even f o r longer pe r i ods . Next morning

they uere r insed in co ld s a l i n e and incubated in the

d e s i r ed s u b s t r a t e .


Tuo methods were used.

l^lodified lead n i t r a t e method

The smears were incubated f o r h in the

f o l l o w i n g s o l u t i o n :

2 v o l s 1% sodium y8-qlycerophosphate

1 v o l 0 .1 a ce ta t e b u f f e r (pH 5 -6 )

1 v o l 2% l ead ace ta te

0.3 v o l 1-5% ngCl2

They uere then r insed in d i s t i l l e d uater and

deve loped in ammoniacal s i l v e r n i t r a t e s o l u t i o n f o r

30 minutes (28% am-nonia uater was added drop by drop

t o 5^ aqueous AgNO^ u n t i l l the p r e c i p i t a t e jus t d i s so l v e s - )

and r insed in 5% sodium th i osu lpha te f o r 5 minutes and

mounted in g l y c e r i n e j e l l y .

m

( 2 ) Coupl ing azo-dye method using hexazo t i z ed p a r a r o s a n i l i n coupler

The f i x e d smears of m i c r o f i l a r i a e were incubated

in a mixture con ta in ing 10 mg c<-naphthyl phosphate in

10 ml of ace ta te b u f f e r . To uhich 750 mg of p o l y v i n y l

p y r r o l i d o n K60 was added and s t i r r e d u e l l t i l l i t became

homogeneous. To t h i s l.n ml o f a mixture con ta in ing

0.5 ml NaN02 (4%) + 0.5 ml HPR uas added and s t i r r e d .

Th i s mixture uas a l lowed to stand be f o r e being added to

the incubat ion mixture . The pH uas ad justed to 5. The

incubat ion mixture having t h i s s o l u t i o n uas f i l t e r e d

d i r e c t l y on to the smears and incubated f o r 30 minutes

at 37 C. The smears were uashed in running uater f o r

2 minutes and mounted in g l y c e r i n e j e l l y .

phosphatase

Only one method i . e . , mod i f i ed coupl ing azo-dye

method uas used to demonstrate the a l k a l i n e phosphatase.

The smears uere incubated in a mixture made by

d i s s o l v i n g 15 mg (10-20 range ) sodium o^-naphthyl

phosphate in 20 ml of 0.1 H stock ' t r i s ^ b u f f e r (pH 10 ) .

To t h i s 20 mg of the s t ab l e d i a z o t a t e o f 5 - ch l o r o -G -

t o l u i d i n e ( s a l t 9 Table 55 Pearse (1968) Diazomium

s a l t s in Enzyme H i s t o chemis t r y ) uas added and s t i r r e d

u e l l . I t uas f i l t e r e d d i r e c t l y on to the smears in a

s u f f i c i e n t quan t i t y to cover them at 20 C in a BOD

70

incubator f o r 15-60 minutes. A f t e r that they uere

washed in running water f o r 1-3 minutes and mounted

in g l y c e r i n e j e l l y .


The smears uure sub j ec t ed to incubat ion f o r 3 h

at 37 C in a b s o l u t e l y f r e sh medium con ta in ing 20 ml of

0,1 n-sodium ba rb i tu ra t e (2.062 g/100 m l ) ; 10 ml of

0.18 n CaCl^ (1 . 998 g/100 m l ) ; 30 ml of d i s t i l l e d water

and 152 mg of adenosine t r iphosphate (disodium s a l t ) .

As soon as ATP was d i s s o l v e d , the pH was ad jus ted to

9.4 with 0.1 n NaOH and the mixture was brought to

100 ml with d i s t i l l e d water . I t was f i l t e r e d , i f

t u r b i d .

A f t e r the incubat ion i s complete the smears wore

washed in 3 changes of CaCl2, t r a n s f e r r e d to 2% C0CI2

f o r 3 minutes, deve loped in d i l u t e y e l l ow ammonium

su lph id e , washed thorough ly , dehydrated , c l e a r ed and

mounted in DPX.

( d ) Carboxy l es t e rase

The smears were incubated at 37 C f o r 3 h in a

mixture prepared by d i s s o l v i n g 1,3 mg o f 5-bromoindoxy1

a c e t a t e in 0.1 ml of absolute a l c o h o l and adding to i t

2 . 0 ml of 0.1 M t r i s - H C l b u f f e r (pH 6.0 or pH 8 . 5 ) ,

71

1.0 ml of 0.05 n potassium ferricyanidG

1.0 ml of 0.05 n potassium f e r rocyan idc

1.0 ml of 0.1 n CaCl2 and the

volume i s made up to 10 ml u i th d i s t i l l e d ua te r .

A f t e r incubat ion the smears uere r insed in ua t e r ,

deihydrated through e thano l grades and c l eared in 1 part

phunol 3 parts xylune and mounted in DPX.

Another method used f o r carboxy l e s t e rase uas

the HPR method. In th i s the smears uere sub jec ted to

incubat ion in the f o l l o w i n g substrate at pH 6 .5 .

7.5 ml of 0.2 PI phosphate b u f f e r stock s o l u t i o n

2.5 ml d i s t i l l e d uater

0.25 ml substrate so lu t i on (1% c/-naphthyl ace ta te

in ace tone ) .

The mixture uas gent ly shaken and 0.8 ml of HPR

uas added.

This mixture was d i r e c t l y f i l t e r e d on to the

smears and the incubat ion uas ca r r i ed out f o r 30 minutes

at 30 C. The smears uere uashed b r i e f l y in ua t e r ,

dehydrateri in graded ethanols and mounted in DPX.


Fixed smears uere incubated f o r 1 h in f r e s h l y

prepared mixture of

72

^ -hydroxyqu ino l ine sulphate 50 mg

[\laCl 200 mg

l/cronal ace ta t e b u f f e r pH 6.0 10 ml

Hexazonium p a r a r o s a n i l i n ( f r e s h ) 0,6 ml

pH 5 .8 -6 .0 ( ad jus ted u i th NaOH)

The smears were uashcd in d i s t i l l e d u a t c r ,

dehydrated , c l e a r ed and mounted in DPX.

4. I_n \/itro c u l t i v a t i o n

Only m i c r o f i l a r i a e uere c u l t i v a t e d ^ v i t r o .

N ight blood uas taken from the uing v e in of h e a v i l y

i n f e c t e d jung le crows. Heparin was added t o t h i s blood

(10 uni ts/ml ) to prevent c o - a g u l s t i o n . Whenever des i r ed

the i n f e c t e d crow was s a c r i f i c e d , during day, the lungs

were taken out and kept in warm (37 C) hepar in i z ed

R i n g e r ' s s o l u t i o n f o r sometime and m i c r o f i l a r i a e were

taken out from the lungs by t e a s i n g them g e n t l y . A f t e r

15-20 minutes the p i e c e s of lungs were d iscarded and

m i c r o f i l a r i a e were separated from t h i s R i n g e r ' s s o l u t i o n

c o n t a i n i n g lung exudate . The m i c r o f i l a r i a e could a l so

be obta ined in l a r g e numbers from the blood d i r e c t l y

taken from heart wi th the help of a s y r i n g e .

Be fore c a r r y i ng out v i t r o c u l t i v a t i o n the

concent ra ted y i e l d s of m i c r o f i l a r i a e were obta ined by

s epa ra t i ng them from blood c o rpusc l e s . This could be

done by;

1. Conglomerat ing RBCs

2. Lysing RBCs

D i f f e r e n t reagents were used f o r ob ta in ing the

m i c r o f i l a r i a e from the b lood .

1. Phytohaemagglut in in

Liith phytohaemagglut inin the RBCs get conglomerated

and s e t t l e down qu i ck l y l e a v i n g m i c r o f i l a r i a e f r e e in

74

plasma. 50 mg phytohaemagglut in in ( D i f c o ) was d i s s o l v e d

in 5 ml of d i s t i l l e d u a t e r . 1 ml of t h i s s o l u t i o n

uas mixed with 9 ml of d i s t i l l e d ua t e r . This uas used

as a stock s o l u t i o n . 0.2 ml of t h i s s o l u t i o n uas added

t o 0.5 ml of b lood and the tube uas gen t l y r o t a t e d f o r

2 minutes f o r mixing p rope r l y the phytohaemagglut in in

u i th the b lood. To t h i s tube 5 ml of R i n g e r ' s s o l u t i o n

uas added and c e n t r i f u g e d at 1000 rpm f o r 3 minutes.

The RBCs s e t t l e d down in the bottom and the supernatant

c on ta in ing m i c r o f i l a r i a e was c a r e f u l l y t r a n s f e r r e d t o

a c e n t r i f u g e tube and c e n t r i f u g e d at 1500 rpm f o r

10 minutes. The supernatant uas d iscarded and the

sediment uas uashed t h r i c e u i th R i n g e r ' s s o l u t i o n by

c e n t r i f u g i n g and d i s ca rd ing the supernatant . Thus a l l

the t r a c e s o f phytohaemagglut in in were removed. The

separated m i c r o f i l a r i a e uere examined under the

microscope f o r t h e i r v i a b i l i t y . This uas supposed t o

be the best method as i t gave maximum y i e l d and the

v i s b i l i t y of m i c r o f i l a r i a e uas not a f f e c t e d .

Uith phytohaemagg lut in in , the RBCs get conglomerated

and so they s e t t l e doun qu i ck l y l e a v ing m i c r o f i l a r i a e

f r n e in supernatant . The purpose of adding more R i n g e r ' s

s o l u t i o n immediate ly a f t e r add i t i on of phytohaemagglut in in

uas to prevent s e t t l i n g doun of m i c r o f i l a r i a e which

would have normally happened i f the volume was l e s s .

75

2. Uater

In t h i s method 1P ml of d i s t i l l e d water was added

t o 0.4 ml of i n f e c t e d b lood. I t uias mixed by shaking

g e n t l y and uas a l lowed to stand f o r 2 minutes and

c e n t r i f u g e d at 1500 rpm f o r 8-10 minutes. The

suoernatant was d i s ca rded . The sediment was mixed with

R i n g e r ' s s o lu t i on and washed tw ice by c e n t r i f u g i n g and

d i s c a r d i n g the supernatant . This method did not g i ve

v e r y good y i e l d .

3. Saponin

For l y s i n g RBCs, saponin was found to be very

e f f e c t i v e wi th subsequent t reatment w i th t r y p s i n f o r

g e t t i n g r i d of the remaining c e l l u l a r c o n s t i t u e n t s .

In t h i s method G.5% s o l u t i o n of saponin and 0.1%

s o l u t i o n of t r y p s i n were prepared in normal s a l i n e and

kept in water bath at 36 C. 4.5 ml s o l u t i o n of saponin

was added to 0 .5 ml i n f e c t e d blood and kept at 32 C

f o r 10 mihutes. The contents wore c e n t r i f u g e d at 1500 rpm

f o r a minutes. The supernatant was d iscarded and to

the sediment 8 ml of R i n g e r ' s s o l u t i o n was added,

thorough ly mixed and c e n t r i f u g e d at 1500 rpm f o r

10 minutes. Three such washings were g i ven by c o n t r i f u g i n g

e ve ry t ime at 1500 rpm f o r 10 minutes and d i s ca rd ing

the supernatant . A f t e r the f i n a l wash 4 ml o f 0.1%

t r y p s i n s o l u t i o n in normal s a l i n e was added to the

sed iment , mixed w e l l , incubated f o r 5 minutes at room

76

temperature and c e n t r i f u g e d at 1500 rpm f o r 10 minutes.

The supernatant uas p i p e t t e d out and the sediment uas

uashed t h r i c c with Rinq.'.r 's s o l u t i o n .

Somotimes f i r s t t reatment f a i l e d to lyso a l l

the RBCs in uhich ease a second treatment wi th Q.2%

saponin s o lu t i on uas g iven f o r 2 minutes.

Saponin uas found to be v e r y e f f e c t i v e u i th

subsequent trf.atment u i th t r yps in f o r g e t t i n g r i d of

remaining c e l l u l a r c o n s t i t u e n t s . This method gave

good y i e l d of m i c r o f i l a r i a e but saponin i s found to be

t o x i c t o the m i c r o f i l a r i a e . Since the v i a b i l i t y of

m i c r o f i l a r i a e i s a f f e c t e d , t h i s method did not prove

t o be good f o r ^ v i t r o cu l tu re as the m i c r o f i l a r i a e

did not surv i ve even f o r normal pe r i ods of t ime .

While using t h i s method another problem arose

due to the nuc lea ted RBCs of crou. A f t e r t reatment

with saponin uhen the blood uas c e n t r i f u g e d the

m i c r o f i l a r i a e uere found to be entang led in a mass

having nuc le i o f RBC. Tryps in could not remove t h i s

complete l y .

A number of exper iments uere per formed to

c u l t i v a t e ^ v i t r o the m i c r o f i l a r i a e of Chandlere11a

hsuking i and induce t h e i r development. (*1any s a l t

s o l u t i o n s , chemica l l y d e f i n ed media and b i o l o g i c a l

supplements uere used. The m i c r o f i l a r i a e uere

separa ted from blood and conc rn t ra t ed . This concentratcr i

77

y i e l d of m i c r o f i l a r i a e uas then dispensed i n t o a s e r i e s

of cu l ture v i a l s having d i f f e r e n t media u i th a n t i b i o t i c s

compris ing 100 units p ^ ' n i c i l l i n and SOyug s t r ep tomyc in .

The cu l ture tubes uere then incubated at 22 C in a BOD

incuba t o r .

A l l g lassware were heat s t e r i l i s e d . The bas ic

s a l t s o l u t i o n s uore s t e r i l i z e d at 15 lb pressuru in an

a u t o c l a v e . The b i o l o g i c a l supplements v i z . , serum, CEE

and de f i ned media NCTC 109, E a g l e ' s PIEM, Medium 199,

Cr-IRL 1066 and Grace ' s i n s e c t t i s sue cu l ture medium GP1A

uere s t e r i l i z e d by f i l t e r a t i o n in S e i t z or P l i l l i p o r e

f i l t e r s . The serum u?s i n a c t i v a t e d at 56 C f o r 1 h in

a water b^th be f o r e use. The exper iments uere c a r r i e d

out under axenic c o n d i t i o n s . The media uere not

changed throughout the cu l ture pe r i od as there uas

e v e r y p o s s i b i l i t y o f mort'^.lity and damage to the

m i c r o f i l a r i a e uh i l e c e n t r i f u g i n g and washing due tn

t h e i r d e l i c r t e s t r u c t u r e . P number of media and

supplements uere usud f o r in v i t r o c u l t i v a t i o n .

78

RESULTS AND DISCUSSION

1. (Morphology


( i ) Adult

Chand le re l l a haukingi C h a t t e r j e e , Sen and

Bhst tacharya , 1965.

The body i s e l onga ted u i th at tenuated ends. The

c u t i c l e appears smooth and devo id o f s t r i a t i o n s under

the l i g h t microscope. The mouth i s t e r m i n a l , s l i t - l i k e

w i thout l i p s . There are tuo pa i r s of c epha l i c p a p i l l a e ,

one pa i r sub-uent ra l and the o ther sub -do rsa l . A pa i r

o f amphids are p r esen t , one on each l a t e r a l s i d e .

Plale

The male i s smal l e r than the f ema l e , 11.85-14.21 mm

in l ength and 0,175-0,212 mm in w id th . The c e p h a l i c end

i s b l u n t l y po in ted . The nerue r i n g i s present at

0.137-0.187 mm from the a n t e r i o r end o f the body and

around muscular part of oesophagus. The e x c r e t o r y pore

i s present 0.112-0,125 mm from the a n t e r i o r end of the

body.

The p o s t e r i o r e x t r em i t y i s blunt and curved

making tuo to three c o i l s . There are f i v e pa i r s o f

p o s t - c l o a c a l p a p i l l a e . The s p i c u l e s are sub-equal and

d i s s i m i l a r u i th t runcated prox imal ends and rounded t i p s .

The l e f t sp i cu l e measures 0.059-0, 072 mm and the r i g h t

0.048-0.062 mm in l eng th .

79

The mouth liBads i n t o the oesophagus uhich i s

d i v i s i b l e i n t o tuo p a r t s : a short a n t e r i o r muscular

par t and a long p o s t e r i o r g landular p a r t . The former

measures 0.125-0,212 mm uh i l e the l a t t e r 0 .46-0 .61 mm.

The tuo par ts t o g e the r measure 0. 59-0. 82 mm in l eng th .

The i n t e s t i n e runs in a s t r a i g h t course and cont inues

i n t o rectum. The c l o a c a l aperture i s present at

0 .175-0 .18 mm from the t i p of t a i l .

The t e s t i s i s convo luted occupying approx imate ly

tuo t h i r d s of the body from p o s t e r i o r end, ex tend ing

a n t e r i o r l y up to the middle o f the g landular part of

oesophagus. The uas de f e r ens i s c o i l e d , f o l l o w e d by

v e s i c u l a semina l i s which g radua l l y widens and f i n a l l y

narrows down forming e j a c u l a t o r y duct which opens in t o

the rectum forming common c l oa ca .

Female

The female i s longer than the male, 15.86-21.19 mm

in length and 0 .24-0.31 mm in width . Both the

e x t r e m i t i e s o f body are b lun t l y po in t ed . The nerve

r i n g i s present around muscular po r t i on of oesophagus,

at 0 .12-0 .15 mm from the a n t e r i o r end of body. The

e x c r e t o r y pore i s l o ca t ed at 0 .125-0 .20 mm from

a n t e r i o r end of the body.

The mouth opens i n t o the oesophagus which i s

d i v i s i b l e i n t o two p a r t s , the muscular part 0 .125-0.275 mm

and the g landular part 0 .4 -0 .44 mm, the two par t s

80

t o g e t h e r measure 0 .5 -0 .55 mm. The i n t e s t i n e does not

open to the o u t s i d e . The anal aperture i s a t roph ied

and represented by a depress i on , at 0 .35-0,37 mm from

the p o s t e r i o r end.

The o v a r i e s are p a r a l l e l , o p i s t h o d e I p h i c ; the

tuio u t e r i run s ide by s ide in a convo luted uay and

un i t e t o form a common vag ina . The v/ulva i s present

at 0 .30-0 .50 mm from the a n t e r i o r end of body,

l/ iv iparous , the uterus i s f u l l of deve loped

m i c r o f i l a r i a e .

B1

( i i ) n i c r o f i l a r j a

Nuclear structures

The m i c r o f i l a r i a e measuring 0.142~0.164 by

n,005 mrn are couered by a d e l i c a t e sheath uhich has

been s ta ined by 3 1C i 3 n blus at pH 5 .0 . The body i s

f u l l of nuc l e i but a smal l a n t e r i o r part devo id of

n u c l e i , is known as c epha l i c snoce , r^easurinq

0 .004-0.005 mm in l eng th . The nerue r ing i s present

at 0 .04-0 .05 mm and the e x c r e t o r y pore at 0 .05 -0 .06 mm

from a n t e r i o r end of the body.

The p o s t e r i o r end of m i c r o f i l a r i a conta ins a

s e r i e s of 4 c e l l s and anal v e s i c l e . The f i r s t i s G

c e l l having l a r ge vacuolated nucleus with nuc l e o lu s ,

present at 0 .042-0.044 mm from Dosterior end of the

body and the r e s t are R^ , R. and R^ c e l l s .

The anal v e s i c l e i s seen behind H - c e l l s , present

at 0.016-0.01R mm frnm p o s t e r i o r end o f the body. The

p a i r e d organs seen betuieen thn t i p of t a i l and anal

v e s i c l e are known as Schuanznebi Ide ui'th separa te ducts

opening o u t s i d e , s i t u a t e d at n.n0'3-0.nn4 mm from

poster i i : ) r end of the body. These are pernaps sensory

in f u n c t i o n . The nuclear column cont inues up to the

t i p of t a i l and there i s a s i n a l e nucleus near the

t i p f F i g . 1 ) .

82

Cephal ic s t r u c t u r e s , oharynqeal thread and Innenkorper

The c epha l i c s t ruc tu res in the m i c r o f i l a r i a of

Chand le re l l a haukinqi have been s tud ied with a v i eu

that they are c h a r a c t e r i s t i c of the genera of f i l a r i a l

uorms and t h i s uould help in i d e n t i f i c a t i o n of d i f f e r e n t

genera from m i c r o f i l a r i a e a lone .

Resu l t s

'3f the dyes used a f t e r o x i d a t i o n , aldehyde

fuchs in gave the best r e s u l t s . Liith t h i s s t a i n , at the

t i p o f c epha l i c space, a hook was d i s c e r n i b l e uhich i s

curved at i t s d i s t a l end and b i f u r c a t e d at the base.

iMear the hook there are four smal l t oo th l i k e s t r u c t u r e s ,

the sp ines , arranged in a t r ansve rse rou uhich look

l i k e equi lat'-^'r a l t r a i n g l e s ( F i q . ? ) . There i s a l so

a u e l l d e f ined r i n g l i k e s t ruc tu r e - the o r a l r i n g .

From near the cent re of t h i s r i n g the oharynqeal

thread s t a r t s as a funne l shaped s t r u c t u r e . I t runs

in t e rne t l l y and r e n t r a l l y to end in the Innenkoroor uhich

i s s i t u a t e d in the a n t f r i ir nart of the p o s t e r i o r t h i r d

o f the body of the m i c r o f i l a r i a . The Innenkorper i s

rouqhly r ec tangu lar in shape. The c u t i c l e and the

sheath could a l s o be d e f i n ed very c l e a r l y . The sheath

looks l i k e a smooth cove r ing around the body of the

m i c r o f i l a r i a . The space betueen the c u t i c l e and the

3 he ath remains almost uniform throughout the body

83

l eng th except at the p o s t e r i o r end uhere i t i s s t r e t ched

apa r t . In t h i s r ep i on , te tueen the c u t i c l e and sheath,

some s ta ined o b j e c t s could be seen. The c u t i c l e i s

t r a n s v e r s e l y s t r i a t e d a l l over the body and at the

p o s t e r i o r end th i cken ing of a Feu c u t i c u l a r s t r i a t i o n s

i s observ/ed. A spine i s seen jn the c u t i c l e at the

t i p of the p o s t e r i o r end ' 'F igs . 3 I 4 ) .

Other dyes l i k e bas ic f u chs in , c r y s t a l v i o l e t ,

b r i l l i a n t c r e s y l b lue , neu t ra l red and a l c i an blue

s t a in ed the hook and spines e qua l l y w e l l but none of

them gave a c l e a r d i f f e r e n t i a t i o n o f the pharyngeal

th r ead . The Innenkorper could be s ta ined w e l l wi th

bas i c f u chs in , c r y s t a l v i o l e t and a l c i a n b lue .

Aldehyde fuchs in a lso gave f a i r l y good r e s u l t s

a f t e r su lphat ion but the s t ruc tu r e s present at the

a n t e r i o r end of the c epha l i c space, i . e . , the hook,

sp ines and thn o r a l r ing could not be d i s t i nqu i shed

SGparatf l y . The t i p nf the c epha l i c space took f a i r l y

qood s tn in u i th othor dyes l i k e bas ic fuchs in , c r y s t a l

v i o l e t and b r i l l i a n t c r e s y l b lue . A f t e r su lphat ion

pharynncal thread could be s t a ined only u i th aldehyde

fuchs in . Innenkorper urns i n t e n s e l y s ta ined u i th

aldehyde fuchs in , bas ic f u chs in , c r y s t a l v i o l e t ,

b r i l l i a n t r resy 1 blut^, n t u t r a l rod and a l c i an b lue .

Sheath could be s ta ined u e l l u i th a l c i a n blue and

bas i c fuchs in but not u i th other dyes. C u t i c l e could

84

be d e f i n ed u e l l only when s ta ined u i th aldehyde fuchs in

and bas ic fuchs in . Comparative e f f i c a c y of d i f f e r e n t

s t a i n s a f t e r o x i d a t i o n and sulphaMon has beon

summarised in Table 1.

D iscuss ion

The s t a i n i n g r oac t i ons of the m i c n f i J a r i a e of

Chand l e r e l l a haukinoi are qu i t e s i m i l a r to those of

Brugia (Laurence and Simpson, 1969) u i th some

d i f f e r e n c e s . A f t e r ox id at i on , aldehyde fuchs in q?\yo

almost e qua l l y good r e s u l t s in both ncratodns in

d i f f e r e n t i a t i n g the s t ruc tu r e s in the c epha l i c space

the Innenkorper , the pharyngeal thread , c u t i c l e and

sheath. With bas ic fuchs in f a i r s t a i n i n g of the

c u t i c l e and sheath could be achieved in case of

m i c r o f i l a r i a of Chand le re l l a haukinqi but not u i th

m i c r o f i l a r i a of Brugia, 'Staining r e a c t i on to c r y s t a l

v/iolet ujas s i m i l a r in both the s p e c i e s , though uary inn

in i n t e n s i t y . Uith b r i l l i a n t c r e s y l b lue , neu t ra l red

and a l c i a n blue thpre uas not -^uch d i f f e r e n c e butucjen

the tuo s p e c i e s .

A f t e r su lphat ion a l s o , aldehyde fuchs in gave

the best r e s u l t s . I t s ta ined the c u t i c l e of m i c r o f i l a r i a

o f Chand le re l l a haukinqi mirn i n t e n s e l y than that Tf

Erug ia . But u i th bas i c fuchs in r e s u l t s were not as

ttD

qood as observed by Laurence and Simpson (1969) in

Bruq ia . Uith c r y s t a l v i o l e t , Laurence and Simpson

(1969) r epo r t ed in t ense s t a i n i n g of nharyngea l thread

and poor s t a i n i n g of c e p h a l i c s t r u c t u r e s . But i t has

been j u s t the oppos i t e in ease of C h a n d l r e l l a .

Laurence and Simpson (1Q69) r e p o r t e d in t ense s t a i n i n o

o f c e p h a l i c s t r u c t u r e s and pharyngea l thread wi th

b r i l l i a n t c r e s y l blue but i t uas not so obvious u i t h

C h a n d l e r e l l a . Hinor d i s s i m i l a r i t i e s were a l s o observed

w i th neu t ra l red and a l c i a n b lue . Accord ing t o o r e sen t

e v a l u a t i o n of the s t a i n s s t u d i e d , aldehyde fu chs in

a f t e r o x i d a t i o n seems to be the best f o r r ou t i n e

examinat ion of m i c r o f i l a r i a e in smears.

Usino the d i f f e r e n t s t a i n i n g t e c h n i q u e s , i t has

been p o s s i b l e to study in the m i c r o f i l a r i a o f C h a n d l e r e l l a

haukinqi some important s t r u c t u r e s uhich sbp'v t o

d i f f e r , from s n e c i e s t o s p e c i e s , in t h e i r charc-'ctor

and arrangement. Table 2 g i v e s a compamt i ve summary

o f the d i s t i n q u i s h i n g cha rac t e r s of these s t r u c t u r e s

in m i c r o f i l a r i a o f d i f f e r e n t s p e c i e s of f i l a r i a e so

f a r s tud i ed by Laurence and Simpson (1169 ) . Fasud on

t h i s a key t o a t i n t a t i v e c l a s s i f i c a t i o n of f i l a r i a e

i s appended be lou . Using ^he key i t i s honed that i-he

common spe c i e s of f i l a r i a e could be i d e n t i f i e d , in the

absence of adul t uorns, from m i c r o f i l a r i a e alonn and

uould be u s e f u l in d i a gnos i s e s o e C i a l l y in cases of

z o o n o s i s .

86

Key to the genera of f i l a r i a l uorms based upon c e p h a l i c s t ruc tu r e s

1. Hook b i f u r c a t e at the base u i th sp ines

Hook not b i f u r c a t e , sp ines absent

2. Hook u i th t ransve rse support ing bars

Hook without support ing bars

3* Hook U-shaped

Spines 4, t r i a n g u l a r

Spines 3, t r i a n g u l a r

Spines 3, f a n g - l i k e

2

5

3

A

Uucherer i a

Brugia p a t e i

B, malayi and

B. pahangi

Spines numerous, arranged

i r r e g u l a r l y in 2-3 rous

4. Hook f a n g - l i k e , sp ines 4,

t r i a n g u l a r in shape

5. Hook u i th tuo t r ansve r se suppor t ing bars

Hook without t r ansve r se bars

6. Hook bou-shaped

7. Hook broad p l a t e l i k e u i th c e n t r a l

p o s t e r i o r p r o j e c t i o n

Hook smal l t oo th l i k e s t ruc ture

Hook s t i l l smal l e r

Loa

Chandlerc11a

6

7

C a r d i o f i l a r i a

Onchocerca

f^lansone 11a

Dipetolonema

Hook beak l i k e L i tomoso ides

87

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(-1 XJ CJ D (D ID a o •P Cb X} 3 * ^ tn CO p n p P U- -p

c c QJ U) 4-) 3 3 T3 •H CD O P u) a> o tn (-1 - O 0! "C U JJ o cn

TJ M c n •n P Q QJ 1 C cn a p ffl -p cu CD XD c: •P 03 4-> £ a 0! cn a a cn a CO u x: P E P 3 0) (0 -p CD tn CD CO 3 1 CO o O • ro m CD •p -C c c o x : Q CD •p I CL CT, jC C a cn 3 o (0 Q) cn r-l p e J LX u QJ S) CO Q) 1 r-H 'rA 1 (D CO CD o 3 > - p C P p •p

O-•P CI XI .IJ cn CD cn X3 to CO d

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91

n j-j

c o u

IN OJ XI TO

(T) w c •H C <n

JSf o o X

0} CO x: D 4-; CU ai

rH CXI XJ o TO E O "D w c CO CD Q) C Q) X e: CD GU c X ai rH Jir cn t-i QJ CD o X cn cn C CO c -o Ci_ cnx f-i r-i f-i cnx X •H CD a CD >- (C c -•J D D Ct~ c u o rH ( - ) X U 03 CO •H •r-i -i-i c •H c T) D 03 CD (-1 TJ M "C (-1 0) cn •rH ,—1 ffi T ) X CD X X c c CD X o fC •a c CO CJ X X C J CD •H X tn f-H cn X •H C.) D • H X

-P CU m •H CD r o D CD t-i f-t cn X P OJ a tt- (-1 c CJ O X X X c llJ O . X D o cC o CD o CJ cn 1— cn •H

ro CD t rH

CT r-i 1 X rH C m r. i •D X' CD OJ -H 1 u ca CD c ( - (

a CO 3 X r. n 03 i-H CU X rH X

( c •a 3 ( (-< a r XJ c K •r-1 c U ( (D E CD • rH C a' (-1 ra (i cc D O (-1 CD CO

(0 X •r-t F CD -t-i X CO X X cz u u CO CO Ct <—1 -X

c a in XI et

W y

4J C (D (n X ci

a.

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-p c CD to X u

•rH X C CJ 1—I 1—1 X o ••H

CD n •H rH (-1 •H G) o X 03 X X X o X rH CD r CD X CD CO rH 1—1 a, ••-> c m (U CO CO a u o r o -H fH E CD

•rH u D rH D cn X rv CD o X X u 0) •—1 u X ' u rH Q n X X CD rH n X D rH H n -):> cn CD (-1 o U •H CD 1-4 H n • H G -X O X x-* X CL 3 Q H 'H '0 V cn cn tn

IQ s a3

CD "0 1 1 ; 1 •D u - i C •rJ M n o ai ' I i CO u c r o n 4-' c_ X cn O u c a X r (T' "H •rH o n X

F i g . 1. NucJear s t ruc tu res of n i c r o f 1 l a r i a .

F in . 2. ucphal ir 3tructij"^os jF m ic rD f i 1 ar ia ,

92

E E

F i g . 1

o V

/

fP h h - s

o V

f i g . 2

Fiq . 3. ] r a l r i n g , nharynqeal th read , Innenkorper

and t a i l spine in long Trm of

m i c n f i l a r i a .

F in. "ja-c 3tructurr js in shnrt form of

n i L- n f i 1 r, I i a .

93

( ^ i g . 3

T i g . 4

94

Forms of m ic ro f i l a r i a s

Uhile studying the smears for m i c r o f i l a r i a e , tuo

forma of m ic ro f i l a r i ae uare observ/ed in C, haukingi.

The long and the short forms^ Both tha forma showed

i d e n t i c a l features . The sheath, the cut i c l e , the hook

and spines, o ra l r ing , pharyngeal thread and Innenkorper

uere a l l i d en t i c a l . The extent of pharyngeal thread,

the pos i t ion , shape and size of Innenkorper i s s imi lar

in re lat ion to the body s i z e . The long forms of

m i c r o f i l a r i a e , measuring 136.4-174.9 j j , are found in

comparatively younger crous and the short forms

72.6-94. 6 yu in older crous. Besides these long and tha

short forms of m i c r o f i l a r i a e , a feu intermediate forms

are also ava i l ab l e . The size of Innenkorper, the

storehouse of mucopolysaccharides, also decreases uith

the shortening of the body ( r i g s . 5 & 6 and Tables 3 & 4 ) .

Further, to v e r i f y that these tuo forms belong

to one species , a number of short , intermediate and

long f':irms have been measured, the posit ion of

Innenkorper has been noted and the tuo r a t i o s , between

the body length and distance of Innenkorper from

anter ior end as wel l as between the body length and

distance of Innenkorper from poster ior end, have been

uorked out. I t i s found that these ra t ios remain

almost constant^Table 5 ) .

95

Discussion

In spite Tf the fact that the body length changes

uith the change in the size of Innenkorper, the tuo

forms of mic ro f i l a r i ae show constant oosit ion of

Innenkorper. The nuclear landmarks and the cephalic

structures are s imi lar in both long and short forms.

These observations go to prove that the size of

m i c ro f i l a r i a e decreases uith the decrease in s ize of

Innenkorper - the storehouse of mucopolysaccharides,

due to the immune responses of host. As the in fect ion

gets o lder , the concentration of antigens increases in

the blood and the m ic ro f i l a r i a e do not get proper food

from thei r surroundings. Their size decreases uith the

decrease in size of Innenkorper as m ic ro f i l a r i a e use up

the stored glycogen from i t for their su rv iva l .

Table Length of m i c r o f i l a r i a e , size and p o s i t i o n of" Innonkorper.

96

S. Len th f .S i ze of ,Oistanco of . -Microf i lar ia . T nnenkomer , Innenkorper

• L e n n t h ' B r e a d t h ' a n t e r i o r

1 72. 6 2.2 2.2 52. 8

2 74. 8 2.2 3. 3 56. 1

3 75. 9 4.4 2,2 57.2

4 R2, 5 2.2 2.2 62.7

5 B5. 8 2.2 3.3 71.5

6 85. 8 2.2 3. 3 72. 6

7 86. 9 2.? ''..3 63. 8

3 86. 9 3.3 3.3 58.3

9 88, 0 2.2 3.3 62.7

10 91. 3 7.2 2.2 51 .7

1 1 91 . 3 2.2 3. 3 67. 1

12 12. a 2.2 68,2

13 5 2.2 " . 3 69. 3

14 93. 5 2.2 3.3 57,2

1 5 94. F 1. 1 3 71.5

1 6 94. 6 2.2 3.3 69. 3

17 94. 6 1 . 1 3. 3 DO. 5

1 R 102.3 2.2 3. 3 77.0

1 9 103.4 3, 3 3.3 74. 8

20 n n . o ^.3 3.3 81 .4

21 11 2.2 3. 84.7

contH. on next page

Table 3 ' contr i . )

S .N. Length of ' " ' i i c r o f i l a r i a

' (

t J i z e o f t i lnncnkorpar t

P i s tancs of Innenkorper

'Lenqth'Brparth, I t 1 > 1

from antt^rior end

22 113.3 3.3 3.3 80.3

23 117.7 4.4 4.4 77.0

24 118.8 2.2 2.2 74. S

25 121.0 3.3 4.4 80. 3

26 122. 1 3. 3 3. 3 80.3

27 124. 3 3.3 3.3 78.1

28 124. 3 3.3 3.3 84.7

29 124. 3 4.4 4.4 85. 8

30 125.4 3.3 3 .3 83. 6

31 125.4 3.3 3.3 85. 8

32 126. 5 3.3 3 .3 88. 0

33 127. 6 3.3 4.4 83. 6

34 130. 9 3. 3 4.4 85. 8

35 130. 9 3.3 3. 3 88. 0

36 134.2 3. 3 3.3 88.0

37 135. 3 3.3 4.4 3R.0

38 135. 3 3.3 4.4 94. 6

39 135.3 3.3 3. 3 94. 6

4 0 136.4 4.4 4.4 97. 9

41 136.4 3.3 4.4 92.4

42 137. 5 4.4 3.3 94.6

43 137.5 3.3 4.4 90.2

nontd. on next page

Table 3 ( contd . )

88

S.N.

I

, Length of , 3 i2e of JInnenkorper

t ,Distance of , Innenkorper

f^icrof i l a r i a 1

I Lfjngfeh i Bre adth I t

( from a n t e r i o r , end

44 138.6 2.2 4.4 91 .3

45 13R. 6 3.3 3.3 93.5

46 139,7 3.3 3.3 92*4

47 139.7 4.4 3. 3 93.5

48 un. B 4.4 3. 3 92.4

49 140, 3 3. 3 3.3 9 6. 8

50 140. 8 3.3 3.3 95.7

51 140. 8 3.3 3.3 99. 0

52 140. 8 3. 3 4.4 91 .3

51 141.9 3. 3 3.3 92.4

54 141 . 9 3.3 3.3 95.7

55 141 . 9 3. 3 10B.9

56 143. 0 3. 3 3-3 96. 8

57 143. 0 3.3 4.4 90.2

58 143.0 3.3 3.3 99, 0

59 144. 1 3,3 4.4 1 01 .2

60 145.2 3. 3 3.3 94. 6

61 145.2 4.4 4.4 99. 0

62 145.2 3.3 3. 3 105. 6

63 145.2 3.3 3.3 99, 0

64 147.4 2.2 2.2 101 ,2

65 148.5 3.3 3 .3 1 04, 5

contd. on n^^xt pagB

T ah 1G 3 ( c a n t d . )

99

, 3 i z e of ,Distance o f 3. N. , Length of , Innenkorper ,Inne nkorpe r

, l^icrof i l a r i a ' Length 'Breadth T f

,frnm a n t e r i o r , 8 nd

66 149. 6 3.3 3.3 102.3

67 149. 6 5.5 4.4 102. 3

68 151 . 8 3.3 3 .3 104. 5

69 151 .8 4.4 3.3 104. 5

70 152.9 3.3 4.4 101 .2

71 152. 9 4.4 3.3 105. 6

7 2 152.'^ 3.3 3,3 107. 8

73 152 . 9 3.3 4.4 101.2

74 1 52 . 9 3.3 4.4 102. 3

75 152.9 4.4 4.4 104. 5

76 154. n 4.4 3.3 100.1

77 154. u 3.3 4.4 103.4

78 156.2 3. 3 3, 3 103.4

79 156.2 4.4 3.3 103.4

8 0 157, 3 3.3 4.4 99. 0

81 157. ^ 3.3 3.3 112.2

82 1 58.4 3.3 3.3 103.4

83 158.4 4.4 4.4 106.7

84 15T.5 3.3 3.3 107. 8

85 1 60. 6 3.3 4.4 104. 5

contd. on nfcxt paqc

Tablf-' ' 'contd. )

100

t3izr ' of .Distance of S.N. _ L&ngth of .Innonknrpor . Innenkorper

N i c r o f i l a r i a 'Length 'Breadth , from a n t e r i o r ! 1 ,and

86

37

88

89

90

91

1 62. B

1 63. 9

169.4

1 69.4

170.5

174. 9

4.4

3.3

4.4

3.3

3.3

3.3

4.4

4.4

4.4

3.3

3.3

110. 0

114.4

112.2

122. 1

11 6. 6

114.4

A l l m G n s u r c m e n t s are in p

101

Table 4. Decrease in the s i z e of Innenkorper u i th the shnrtening of the body.

j.!\l. Body lenqth S ize of Innenkorper

1 169.4 4.4 X 4.4

2 158.4 4.4 X 4.4

3 1 56.2 4.4 X 3.3

4 152. 9 4.4 X 3.3

5 151.8 3.3 X 3.3

6 138. 6 3.3 X 3.3

7 135. 3 3.3 X 3.3

8 103.4 3.3 X 3. 3

9 1 n?. 3 2.2 X 3.3

1 0 91 . 3 2.2 X 3. 3

1 1 82. 5 2.2 X 2.2

12 72. 6 2.2 X 2.2

A l l measurements are in P-

102

E o (-1

u 01 a u o ^ c CD c c J—(

o CD o c CD -P tn •H •o •a •

c •D ro C

0 x: •p (H cn O c •H (D -H CD -P

cn n o o Q-X3

cn CD CO JZ -P r-i

r-H c CD CD 3 CD 3 CO -P CO CD XI M

n cn •M n* (-4 •tH CD 4-> -P CO C u:: CO

LO (D JD

CO o -p CO CM n

E P U ci_ C

O- f-i en n CD

Q f-1 Q U O o -.n -w c ^ p CO c Q} -4-) Q) -P cn c CO •H c o C) o

e n M Ct-

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F i g . S. Photomicronraph of long form of

m i c r o f i l a r i a . X 1100.

»1

103

Fig. 6. Photomicrograph of short form of

m i c n P i l a r i a . X noo.

104

Fig. 6

105

( b ) S tereoscan sturi ies

( i ) Adult

The body c u t i c l c has bpRO doscr ibod to bt. Gmooth

by l i g h t microscopy but i t shous s t rong s t r i a t i o n s at

a n t e r i o r and p o s t o r i o r ends of body. The mid rngion

of body shows f e e b l e s t r i a t i o n s . The mouth i s

e l l i p t i c a l , mounted on a r a i s ed s h i e l d of c u t i c l c ,

Nothing s p e c i a l could be observed r egard ing copha l i e

o a p i l l o e and amphids, only that amphids are su^;n to

have a peduncle. The anal depress ion in female i s

found to be surrounded by f e e b l e uauy s t r i a t i o n s u i th

a feu small ua r t s . Wo d i f f e r e n c e was observed in

c e p h a l i c s t ruc tu res of Tiale and female . The t a i l in

f ema le shous s t r i a t i o n s u i th b l u n t l y rounded t i n . The

t a i l of male dot_3 not show any a d d i t i o n a l f e a t u r e s

( F i g s . 7 - 1 3 ) .

( i i ) H i c r o f i l a r i a o

The s te reoscan s tud i es of m i c r o f i l a r i a e did not

r e u e a l anything s o e c i a l as hhey are cov/erud u i th a

sheath on l y ; the sheath shows wr ink l es at th(i p o s t t r i o r

end in long forms and i t i s wr ink led throughout in

shor t forms ( F i g s . 14 & 15) .

F i g . 7. Scanning e l c c t n n micrograph of a n t e r i o r

pnd of adu l t . X 1500.

F i g . 8, Scanning r l e c t n n micrograph of head

en - face uieu of adu l t . X 1000.

F i g . 7

Fig. 8

F i g . 9. Scanning e l e c t r o n micrograph of genera l

c u t i c u l a r surfacFj in midbody reg inn of

adu l t . X 5000.

F i g . 10. Scanning e l e c t r o n micrograph of vu lva

of f emale . X 700.

107

Fig. 9

/ i g . 11. Scanning e l e c t r o n mirrnqraph of c u t i c u l a r

ornamentat ion around anal depress ion

of f emale . X IIOOG.

F i g . 12. Scanning e l e c t r o n micrograph of t a i l

of f emale . X

108

Fig. 11

Fig . 12

F i g . 13, Scanninq e l e c t r o n minrnqraph of t a i l

o f male. X

109

Fig. 13

F i g . 14. ocannin-. e l e c t r o n micrograph of short

form of m i c r o f i l a r i a . X 3100.

F i g . 15. Scannin-'' e l e c t r o n rriicronranh of a n t e r i o r

find of short f i r r of m i c r o f i l a r i a , X 11000.

134

Fig. 14

F i g . 110

I l l

2. Histology

The present uork deals uith the h i s t o l og i c a l

studies of Chandlere11a haukingi, a f i l a r i a l parasite

of Indian jungle c n u Cprvus macrprhynchos (Uag l e r ) .

BODY IxJALL

The body u a l l consists of cu t i c l e , sub -cut ic le

and muscle layer . These layers are very intimately

associated uith each other.

Cuticle

This is outermost, semitransparent covering of

the body. Besides covering the externa l parts i t passes

through a l l apertures l ike mouth, vulva and c loaca,

forming interna l l in ing of oesophagus, vagina and rectum,

The s u p e r f i c i a l layer of cut ic le forms pap i l l a e ,

cephal ic as u e l l as post -c loaca l and s t r i a t i ons on the

body (The cut ic le shous strong s t r i a t i ons in anterior

and poster ior part of the body and feeb le s t r i a t i ons

in the middle region of the body by stereoscan electron

microscopy). In transverse section the cut ic le shows

f i v e layers (F ig . 15 ) . These layers are as f o l l ows :

1. Outer co r t i c a l layer

2. F i b r i l l a r layer 3. Matrix layer 4. Fibrous layer 5. Basement membrane

112

The external co r t i c a l layer is quits dense as

compared to the other layers . Matrix layer appears l ike

a spongy layer . The f i b r i l l a r layer is perhaps formed

by condensation of matrix layer . In this layer the

f i b r i l s form the netuork. The f ibrous layer is made

up of connective t i s sue . The basement membrane is a

thin layer .

These layers arc not present throughout the

body. Only outer co r t i c a l layer is present in the

l in ing of oesophagus v/agina and cloaca. The cut ic lc is

thicker in male than in female, e spec ia l l y in the

greater part of the middle region of body. This may be

due to the enormous development of uter i in female.

Discussion

The cut ic le of the present form resembles the

cut ic l e of S_. corvi as described by Ansari and Basir

(1964) but in _£, haukinqi only 5 layers could be

observed uhereas Anspri and Basir (196A) described

8 layers in cerv i . Goldschmidt (1906) described

9 layers in the cut ic le of Ascaris lumbricoides.

The nature of cut ic le has also been discussed

by some uorkers. Grubbe (1850) termed i t ch i t in .

Flury (1912) ca l led i t keratin and Plagath (1919) termed

i t cut ic le in case of Ascar is . Mueller (1929)

described the cut ic le as a secretion product, but did

not mention the nature of the product. Chituood (1936)

113

and Ansari and Basir (1964) bsliewed i t to be proto-

plasmic condensati-TH from l iv ing c e l l s . The author

a lso agrees with the vieu of Chj.tunod (1 936) and Anaari

and Basir (1964).

Sub-cut ic le

I t i s present belou the c u t i c u l a r l aye r in the

form of 3 f i n e sheet of protoplasm. I t becomes th in

towards both the e x t r e m i t i e s but becomes t h i c k e r in

the middle reg ion of the body in male and in p o s t e r i o r

par t of the a n t e r i o r one t h i r d of the body in f ema le .

In the middle r eg i on of the body of f ema l e , the

s u b - c u t i c l e gets pressed due to the enormous grouth of

u t e r i . This l a y e r bulges i n t o pseudocoelom at four

p laces in the form of l o n g i t u d i n a l chords; one d o r s a l ,

one v e n t r a l and tuo l a t e r a l s , one on e i t h e r s i d e .

The do r sa l chord i s th inner than the v e n t r a l uh i l e the

l a t e r a l s are most prominent. These chords d i v i d e the

musculature i n t o four groups. The nuc l e i are seen

on ly in the chorda l r e g i o n . A l l the four chords

conta in nerve c e l l s and the l a t e r a l ones lodge l a t e r a l

e x c r e t o r y channels a l s o . The chords begin as smal l

th i ckened s t ruc tu res in the a n t e r i o r r e g i on o f the

body where nuc l e i are not seen in i t . These chords

become mod i f i ed at d i f f e r e n t l e v e l s . In oesophageal

r e g i on they become high g i v i n g support t o the oesophagus

by surrounding i t p a r t l y ( F i g . 1 7 ) , These g i v e support

114

t o vu lva in f ema le . In the r eg i on of gonads the chords

become h igh ly f l a t t e n e d . In t a i l r e g i on they become

inconspicuous.

Discuss ion

This form resembles B. oahangi , as desc r ibed by

Schacher (1962) , in having four sub - cu t i cu l a r chords .

Ansar i and Basir (1964) descr ibed four a d d i t i o n a l

r i d g e s , tuo sub-dorsa l and two s u b - v e n t r a l l y i n g on

e i t h e r s ide of l a t e r a l chords in case of c e r v i .

Hany workers have d iscussed i t s o r i g i n a l s o .

Hamann (1895) regarded i t ec todermal whi l e zur Strassen

(1904) b e l i e v e d i t to be mesodermal in o r i g i n . Stewart

(1906) c a l l e d s u b - c u t i c l e as hypodermis. Schacher

(1962) d id not d iscuss i t s o r i g i n .

flUSCULATiJRE

Ins ide the s u b - c u t i c l e l i e s the somat ic

musculature. The four sub - cu t i cu l a r l o n g i t u d i n a l chords

d i v i d e the musculature i n t o four s e c t o r s . The somatic

musculature ge ts mod i f i ed forming s p e c i a l i s e d muscles

in oesophagea l , i n t e s t i n a l , v u l v a r , anal and sp i cu l a r

r e g i o n .

The somatic musculature i s coe lomyar ian ,

polymyarian t y p e , each muscle c e l l c ons i s t s o f a

prox imal f i b r i l l a r part and d i s t a l p ro top lasmic

nuc lea ted pa r t . The f i b r i l l a r part i s at tached to the

115

s u b - c u t i c l e and pro top lasmic par t l i e s Free in the

pseudocoe l ( F i g . The suppor t ing f i b r i l s are

present in o ro top lasmic pa r t . These f i b r i l s have

connect ions u i t h the nervss running i n s i d e the chords.

The s p e c i a l i s e d muscles suppor t ing oesophagus

are knoun as aomato-oesophageal muscles. They extend

from somatic musculature t o oesophagus to giv/e support

t o i t . These muscles are at tached t o the oesophagus

at the lev/el o f or a l i t t l e behind the nerue r i n g . These

muscles do not form any d e f i n i t e pa t t e rn (F igs . 17 ^ 19 ) .

Those suppor t ing the i n t e s t i n e are termt-a .'s

s o m a t o - i n t e s t i n a l muscles. These are in the form of

bands or a sph inc te r around the i n t e s t i n e ( F i g . 2 0 ) . In

male the sp i cu l e s are supported wi th p r o t r a c t o r and

r e t r a c t o r muscles. In f ema l e , these muscles g i v e

support t o uulua and are knoun as somato-vulvar muscles.

D i scuss i m

Uhi le s tudy ing the musculature of nematodes

Schneider (1B60) found that the form, number and

arrangement of muscles are of g r ea t importance in

c l a s s i f i c a t i o n of nematodes. On t h i s bas is he grouped

the nematodes i n t o tuo groups, p la tymyar ian and

coe lomyar ian . The p latymyar ian are the nematodes in

uhich the f i b r i l l a r po r t i on of muscles i s f l a t towards

the body c a v i t y and coelomyarian are those in uhich

the f i b r i l l a r po r t i on of muscles bear groove and the

116

pro top lasmic part dip down in t o the body c a v i t y

( p s e u d o c o e l ) . Further on the bas is o f the number of

muscle c e l l s in each s c c t o r , he suggested tuo groups of

nematodes, mernmyarian hau/ing feu muscle c o l l s in cach

s e c t o r and po lymyar ian, i f they are numerous,

Har t in i (1916) r e s tud i ed the musculature of

nematodes and suggested that polymyarian and coelomyarian

nematodes are raeromyarian and p latymyar ian by f i n d i n g

the t r a n s i t i o n a l forms between the two in the l a r v a l

s t a g e s .

Schachei' (1 962) did not d iscuss t h i s po in t in the

case of Brugia pahangi .

Ansari and Basir (1964) suggested that polymyary

and coelomyary i s reached in l a t e r s tage of devolnprnent.

They descr ibed somatic musculature in S e t a r i a c e r v i as

polymyarian and coe lomyar ian t ype . They a l so desc r ibed

the s p e c i a l i z e d muscles, the somato-oesophagcal muscles,

s o m a t o - i n t e s t i n a l muscles, somato-uulvar muscles,

somato- r e c t u l muscles, musculus an i , copu la to ry

muscles and sp i cu l a r muscles.

The musculature in Chandlerc11a hawkingi i s

almost s i m i l a r t o the musculature of c e r y j on ly wi th

few minor d i f f e r e n c e s . In the former musculus ani

and copu la tory muscles are not observed .

117

BODY CAUITY

The body c a v i t y i s a pseudocoe l , a n t e r i o r l y in

the r eg ion i f muscular oesnphagus and p o s t e r i o r l y in

t a i l r e g i o n , the body cav/ity i s comple t e l y o b l i t e r a t e d

due to the enormous growth of connec t i ve t i s s u e . In

f ema l e , in the middle r eg i on of the body almost a l l the

space i s f i l l e d wi th c o i l e d r ep roduc t i v e organs. The

body f l u i d i s g ranular . Some nuc leated connec t i ve t i s sue

i s a l s o seen in pseudocoe l in oesophageal r e g i o n .

P i s cus s i on

Goldschmidt (1906) desc r ibed the body c a v i t y in

A s c a r i s t.o be surrounded by ' i s o l a t i o n t i s s u e ' uhich i s

a membranous t i s sue support ing va r i ous organs. S teuar t

(1906) found nuc l e i in j e l l y l i k e mass in Oncholaimus

and b e l i e v e d the body c a v i t y to be f i l l e d u i th

mesenchyme but because hn could not see the c e l l u a l l

around the nuc l e i he did not emphasize his op in i on .

Ansari and Basir (1964) gave d i f f e r e n t v iews r ega rd ing

the body f l u i d . They suoqested that the body f l u i d i s

e i t h e r a f l u i d uhich ge ts co -agu la t ed when the worms

are t r e a t b d wi th f i x a t i v e or i t i s a mesenchymatous

t i s s u e of lou o r g a n i s a t i o n . They a l so presumed i t t o be

a f l u i d in p lace of c i r c u l a t o r y f l u i d .

^^ £• haukingi the body f l u i d i s j e l l y l i k e

granular substance u i th some nuc l e i in i t . I t may be a

f l u i d in p lace of c i r c u l a t o r y f l u i d .

118

CQMNECTI\JE TISSUE

I t i s nuc l ea t ed , granular mass of t i s sue present

around the oesophagus. I t i s not seen in any o ther r eg i on

i n t h i s case .

D iscuss ion

The connec t i ve t i s s u e uas g iven d i f f e r e n t names.

Schneider (19Q2) c a l l e d i t " Bindegeuebe" . L o j s s (1905)

named i t " s t r and l i k e organs" . Goldschmidt (1906)

proposed the term " i s o l a t i o n g e u e b e " f o r i t . Ansari and

Bas i r (1964) suggested i t to be l i k e a mesentry ho ld ing

va r i ous i n t e r n a l organs in t h e i r p l a c e . In Chandlere11a

hawkingi t h i s t i s s u e i s l i k e a mesentry and l i m i t e d

only t o oesophageal r e g i o n .

DIGESTIVE SYSTEM

D i g e s t i v e system cons i s t s of mouth, oesophagus,

i n t e s t i n e , rectum, opening out through c l o a c a l aperture

in male and ending b l i n d l y in anal depress ion in ft jmale.

The mouth i s s imp le , present on ra i s ed c u t i c u l a r

p l a t e . I t leads i n t o oesophagus.

Oesophagus

The oesophagus i s d i v i s i b l e i n t o two p a r t s , the

a n t e r i o r muscular and the p o s t e r i o r g l andu la r . I t i s

covered by a th in membrane, the tun i ca p r o p r i a .

119

The muscular part of oesTphagus i s smal l e r and

narrouer than the g landular part of oesophagus. I t s

u a l l s are th ick^ muscular u i th a t r i r a d i a t e lumen. The

lumen i s supposed to be formed of c u t i c l e uhich covers

the b3dy of nematode e x t e r n a l l y . The g landular part o f

oesophagus i s lonqt^r and broader than the muscular part

i f oesophagus. I t s u a l l s are s y n c y t i a l . This part

o f t e n shows s l i g h t l y muscular charac tor around the lumen,

f r i m uhich muscular rays are seen p r o j e c t i n g i n t o

s y n c y t i a l mass. The lumen i s o f t e n c i r c u l a r . The

muscular part of oesophagus i s supported by somato-

oesaphagea l muscles uhich s t a r t from body u a l l

musculature t o g i ve support to oesophagus (F igs , 17 & 19 ) .

The g landular par t of oesophagus i s supported by

sph inc t e r muscles ( F i g . 21 ) .

A nerve r i ng i s seen around the muscular part

o f oesophagus. I t i s present o b l i q u e l y as in s e c t i o n s

i t i s always Sfjen in semilunar form ( F i g . 2 2 ) . Hugo

quan t i t y of connec t i ve t i s sue i s found to f i l l up the

space between the body w a l l and the oesophagus ( F i g . 2 3 ) .

I n t e s t i n e

The oesophagus i s f o l l o w e d by i n t e s t i n e which

i s th in wa l l ed . The u a l l s are made up of a number of

c e l l s and hence i t i s of myr iocytous typs showing 6-10

nuc l e i per s e c t i o n . The c e l l s of the w a l l are of

e p i t h e l i a l type ( F i g . 20 ) .

Re ctum

The i n t e s t i n e becnmes s l i g h t l y wider forming

rectum uhich ends b l i n d l y in an anal d ep r e s s i i n in

female and opens out through c l oaca in male. Rectum i s

l i n e d i n t e r n a l l y by a c u t i c l e which i s a con t inuat i on

of the e x t e r n a l c u t i c l e ,

Cloaca

Cloaca i s a common c a v i t y formed by recturm,

and e j a c u l a t o r y duct opening i n t o i t . I t i s l i n e d

i n t e r n a l l y by c u t i c l e . Sp icu les are o f t e n seen

p r o j e c t i n g through t h i s o p e n i n g ( F i g . 2 7 ) .

Discuss ion

The nature of oesophagus of C. haukingi i s

s i m i l a r t o the oesophagus ] f B_. pahanqi as desc r ibed by

Schacher (1962) and 3. c e r v i Ansari and Basir (1964 ) .

The only d i f f e r e n c e i s that the g landular part of

oesophagus - ' f ten shows a c i r c u l a r lumen ins t ead of a

t r i a n g u l a r one.

The i n t e s t i n e i s a lso of myr iocytous and

an isocy tous type as c a t e g o r i s e d by Chituood (1930 ) .

The w a l l s hav/e l a r g e number of c e l l s and the lumen i s

i r r e g u l a r . pahangi and S. c e r v i a l so f a l l in the

same c a t e g o r y .

121

m i l REPRODUCTII/E SYSTEM

The male r ep roduc t i v e system i s tubular and

r m m r c h i c . The t e s t i s i s thread l i k e and c o i l e d . I t

beg ins u i th ane c e l l in the muscular par t nf oesophagus

and proceeds backuards and becomos s e v e r a l c e l l t h i c k .

The t e s t i s leads i n t o tubular vas de f e r ens of

approx imate ly same width as that of t e s t i s . The

vas de f e r ens i s qu i t e long and leads i n t o u ider seminal

v e s i c l e which i s f o l l o w e d by muscular e j a c u l a t n r y duct .

The e j a c u l a t o r y duct opens i n t o rectum jus t in f r o n t of

the c l o a c a l aper ture . This common tube i s known as the

c l o a c a .

T e s t i s

The t e s t i s c ons i s t s of two p a r t s , the germinal

zone and the growth zone. The germinal zone s t a r t s

wi th one c e l l but the number inc r eases g radua l l y as i t

proceeds p o s t e r i o r l y . This z :no i s covered over by

f l a t c e l l s . The g. rm c e l l s i n s i d e t h i s cove r ing are

compact ( F i g . ) .

The germinal zone passes i n s e n s i b l y i n t o growth

zone . This zone i s a l so coverea over by f l a t c e l l s .

The c e l l l i n i n g i s th in in the a n t e r i o r r e g i on but

becomes th i ck p o s t e r i o r l y . The c e l l s i n s i d e t h i s

c o v e r i n g are known as spermatogonia ( F i g . 2lf ) .

2

l/as de fe rens

The g n u t h z )ne i s f-^llnuod by vas de ferens uhich

i s a lso nf the same width. The us l l a are glandular and

the lumen i s f u l l i f dcuclopinQ spermatoz:)a uhich are

smal l s phe r i c a l s t ruc tures ( F i g . 2 5 ) .

Seminal v e s i c l e

The uas de ferens uidens to form the seminal

v e s i c l e . This part s t o r e s the sperms. The u a l l s are

made up o f f l a t and narrou e p i t h e l i a l c e l l s . The lumbn

i s f u l l of f u l l y developed spermatozoa ( F i g . 26 ) .

E j a cu l a t o r y duct

The seminal v e s i c lQ i s fo l loujod by muscular

e j a c u l a t o r y duct. The outer layer of u a l l s i s of

c i r c u l a r muscles and the inner l ayer i s of columnar

c e l l s . This duct opens in t D common c loaca ( F i g . 2 7 ) .

Sp icu l es

The sp i cu l es are sub-equal , d i s s i m i l a r and

s c l e r ^ t i z e d s t ruc tu r es . They are covered by a sp i cu la r

sheath made of c i r c u l a r muscles and are supported by

sp i cu l a r muscles ( F i g . 2 8 ) .

Discussion

he male reproduct i ve system of C. haukingi

shous characters s im i l a r to that of Brugia pahangi as

descr ibed by Schachcr (1 962) and Se t a r i a cerui by

ftnsari and Basir (1964) .

•e 23

Schacher (1962) and Ansari and Basir (1964)

d e s c r i b ed t e s t i s f o l l o w e d by seminal v e s i c l e but in t h i s

case t e s t i s i s f o l l o u c d by v/as de feruns and the l a t t e r

l eads i n t o wider seminal v/'isiclo which opens i n t o

c l oaca through a muscular e j a c u l a t o r y duct . Cobb (1905)

and Rauther (1909) descr ibed hair l i k e processes in the

lumen of vas de f e r ens but they are ne i the r desc r ibed by

Schacher (1962) and Ansari and Basir (1964) nor they

are seen in C. haukingi .

FEHALE REPRODUCTIVE SYSTEM

The female r ep roduc t i v e system cons i s t s o f pa i r ed

o v a r i e s , o v i duc t s , receptaculum semin i s , u t e r i and a

common uterus , vag ina and vu l va .

Ovary

The j v a r i o s aru d i d o l p h i c and o p i s t h o d e I p h i c .

They are c o i l e d s t r u c t u r e s . Each ovary c ons i s t s of an

outer e p i t h e l i a l l aye r and an inner germinal chord. A

smal l cap c e l l i s present cove r ing the-: b l ind end of

ovary ( F i g . 29) , Each ovary has two d i s t i n c t zones

the germinal z ine and the growth zone.

The germinal zone s t a r t s jus t behind the a p i c a l

c e l l . This area i s the area of rap id d i v i s i o n . I t

s t a r t s as a s i n g l e c e l l but the number inc reases as i t

proceeds backwards. The c e l l s arc s p h e r i c a l w i th a

124

l a r g e nucleus and a nucleo lus ins ide ( f i g . 30 ) . Thyse

c e l l s are enc losed by e p i t h e l i a l l a y e r . This i s fo l louedr-

by the grouth zone .

The grouth zone i s r e l a t i v e l y longer than

germinal zone. The c e l l s f i l l i n g up t h i s part are kniun

as oogon ia . These oogonia are covered i ve r by a

compara t i v e l y t h i c k e r l aye r of e p i t h e l i a l c e l l s ( F i g . 3 0 ) .

Oviduct

Each ovary i s f a l l o w e d by the ov iduc t . The w a l l

o f the ov iduc t c ons i s t s of an outer l a y e r o f c i r c u l a r mus-

c l e s find an inner l a y e r of columnar c e l l s ( F i g . 3 0 ) '

Uterus and receptaculum seminis

The ov iduct of each s ide i s f o l l n u e d by uterus.

At the junc t i on of ov iduct and utorus the tube becomes

a l i t t l e en la rged f :)rmlng receptaculum seminis f o r

s t o rage of sperms. The wa l l s of receptaculum seminis

ore made up of i r r e g u l a r columnar c e l l s with nuc l e i in

the basa l pa r t . The columnar c e l l s are l i ned e x t e r n a l l y

by a membrane of e p i t h e l i a l c e l l s ( F i g , 3I ) . The

receptaculum seminis narrous diun and proceeds as

uterus proper .

The two u t e r i i run a n t e r i o r l y and uni te forming

common uterus. The u a l l s of uterus arc made up of l ou ,

r e c t angu la r e p i t h e l i a l c e l l s . The lumen i s f u l l of

m i c r o f i l a r i a e , in va r i ous s tages of development ( F i g . 3 2 ) .

125

As i t proceeds a n t e r i n r l y i t s u a l l s become mars and

more th i ck forming muscular uagina.

V/sgina

The vag ina c ons i s t s i f tuio p a r t s , the par t c l o se

t o the uterus i s knoun as vag ina u tc r ina and the par t

opening t o the outs ide i s kniun as vag ina ve^ra or

t rue vag ina . The u a l l s of vag ina u te r ina are made of

columnar c e l l s surrounded by c i r c u l a r muscles ( F i g . 21).

The u a l l s of vag ina ve^ra are th i ck wi th an inner l aye r

o f columnar c e l l s and an out^r l a y e r o f c i r c u l a r and

ob l i que muscles (Figs. 32 33 ) .

D iscuss ion

D i f f e r e n t workers gave d i f f e r e n t op in ion r ega rd ing

the o r i g i n and e x i s t e n c e of cap c e l l present at the

b l i nd end of ova ry . Chituood (1 929) descr ibed t h i s c e l l

as a part o f o v a r i a l ep i the l ium. I^usso (1930) gave a

d i f f e r e n t v i ew . He sa id that the cap c e l l i s an

u n d i f f e r e n t i a t e d germinal stem c e l l which p r o l i f e r a t e s

both qerminal and e p i t h e l i a l c e l l s . Ansari and Basir

(1964) found i t underneath the e p i t h e l i a l c e l l s

c o v e r i ng the germinal zone of o va r y . The author

b e l i e v e s i t t o be a c e l l o f d i s t i n c t o r i g i n .

The receptaculum seminis i s another po in t o f

c o n t r o v e r s y . Hagath (1916) b e l i e v e d i t to be a part

of ov iduct whi le Chituood (1933) descr ibed i t as a

126

m o d i f i e d part of utnrus. The author i s a l so of the

op in ion that t h i s part i s a m o d i f i c a t i o n of uterus as

in young specimens t h i s part i s d i s t i n c t l y se t o f f from

the o v a r i e s and u t e r i but in mature females t h i s

c o n s t r i c t i o n i s l o s t and receptaculum seminis becomes

c on f lu en t wi th uterus . This g i v e s an ev idence that

seminal r e c e p t a c l e i s a mod i f i ed par t of uterus.

Fundamentally the female r ep roduc t i v e system

resembles both the p a r a s i t i c f i l a r i a e B. pahangi and

S. c e r v i so f a r h i s t o l o g i c a l l y s t u d i e d .

F i g . 16. T . T . shouino l a y e r s of c u t i c l e of body.

i q . 17. T . 3 . shouinq hioh chords of s u b - c u t i c l e ,

musr^uI'.' r ' "art of oosociha, us nod sotnato-

o-isonhaqpol nus r l e s .

F i g . 15

127

o m

CU.

mus oes. som.oes.mus.

20/U

F i g . 17

12t

F i g . 18

o IT)

muse. mus.oes. lat.ch.

som.oes.mus.

v.ch.

F i g . 19

F"ig, 20. T . S . shouing i n t e s t i n e and somato-

i n t e s t i n a l muscles.

F i g , 21. T .S , shouing g landular part of

oesophagus and u?gina u t e r i n a .

134

o ui

dorsch. cu. muse.

F i g . 20

gl.oes.

som.oes.mus

F i g . 21

F i g . 22. T .S , shouing nerve r ing around muscular

part of oesophagus.

F i g . 23. T .S . shouing ronnec t i v e t i s sue F i l l i n g

up space body w a l l and

oesophagus.

13.0

O m

F ig . 22

to con.t .

F i g . 23

F i g . 24. T .S shouing germinal and growth zones

of t e b t i s .

F i g . 25. T .S . shouing uas d e f e r e n s .

134

s

V. c h .

F iq . 24

O U)

vas.def.

F i g . 25

F iq . 26. T . 5 . showing seminal v e s i c l e ,

F i g . 27. T . 5 . shouinq n l i a c a .

155

o o in in

F iq . 26

o If)

muse. d e j . sp. sp.s. latch. r e c t .

F ig . 28. T . 3 . shouing rectum, ductus e j a c u l a t o r i u s ,

and sp i cu l e s u i th sheath.

F i g . 29. T.- i . shnuiinr cac c u l l of ovary .

157

o lO

F i g . 28

o u>

cap c

F i g . ?g

F i g . 30. T . 3 . shouing /ones of ovary and ov iduct ,

f^ig. T . 5 . shouing rsceptaculum semim's.

134

F ig . 30

.ov. germ z.

ov. gr.7.

oogn.

F i g . 31

F i g . 32. T .S . shouinq uterus .

F i g . 33. T .S . shouinq ur^gina vera,

135

saoi6

F i g . 32

•saoiB

•jaA'6oA

3. H is tochemis t ry

( i ) Adult


( b ) A l ka l i n e phosphatase


( d ) Carboxyl e s t e rase


R e s u l t s :

The c u t i c l e an.j the musculature shou f e e b l y

p o s i t i v e r eac t i on Tor acid ohosphatase a c t i v i t y .

In the a l imentary cana l , the oesophagus, the

i n t e s t i n e and rectum are s t r ong l y p o s i t i v e f o r ac id

phosphatase.

In female reornduct iue system, the germinal and

qrouth zones of oua r i o s shou a moderate ly p o s i t i v e

r e a c t i o n and the e p i t h e l i a l c e l l s c ove r ing the n v f r i e s

shou a s t r o n g l y p o s i t i v e r e a c t i o n . The columnar c e l l s

o f receptaculum seminis shiu a p e cu l i a r type of r eac t i on ,

The e n t i r e c e l l o f the columnar ep i the l ium does not

e x h i b i t the r e a c t i o n but only some l o n g i t u d i n a l strand

l i k e s t ruc tures ins ide the c e l l s shou a p o s i t i v e

r e a c t i o n f o r t h i s enzyme. The wa l l of the u te rus ,

vag ina u te r ina and vanina ve/ra shou s t rong p o s i t i v e

r e a c t i o n .

137

In male, only the a l imentary canal shows a

p o s i t i v e r eac t i on f o r ac id ohosphatase a c t i v i t y ( F i g s . 3 4 - 4 1 )

D iscuss ion

Acid phosphatase has been de t e c t ed h i s t o c h e m i c a l l y

in s i t e s assoc ia t ed with a b s o r p t i v e , s e c r e t o r y and

e x c r e t o r y func t i ons and so a t t e n t i o n has been paid to

the l o c a l i z a t i o n of nhosphatases in r e l a t i o n to these

f u n c t i o n s . For i ns tance , in Hymenolepis diminuta

(Dike and Read, 1971a,b) and Fas c i o l a hepat i ca

(Nansour, 1959; Prober t and Luin, 1974) the tegument

uhich performs abso rp t i v e f u n c t i o n s , shows a s t r o n g l y

p o s i t i v e ac id phosphatase a c t i v i t y . On the o ther hand

in Ascar i s lumbr ico ides nu t r i en t s are absorbed from

the i n t e s t i n e (Sanhueza, P a l r a , Gberhauser, Grrego ,

Parson and S a l i n a s , 1963) , and t h i s organ shous a

s t r ong phos'^hatase a c t i v i t y . In -^any other nematodes,

the lurninal sur face of i n t e s t i n e i s r i ch in ac id

phosphatase i n d i c a t i n g i t s involvRment in absorp t ion of

n u t r i e n t s , as ue11 as s e c r e t o r y f u n c t i o n s .

The hypodprmal ac id phosphatase has been

demonstrated h i s t o chem i ca l l y in doo heart worm,

D i r o f i l a r i a immit is fYanaqisaua and Koyama, 1970) .

S i m i l a r l y in C. haukini^i, a p o s i t i v e r e a c t i o n

in a l imentary canal proyeg that i t i s a c t i v e l y i n vo l v ed

in abso rp t i v e and s e c r e t o r y f unc t i ons .

138

The g landular sur f aces of female r ep roduc t i v e

system shou a p o s i t i v e r e a c t i on and thus i t apnears

tha t ac id phosphntase i s a s soc i a t ed with s e c r e t o r y

f u n c t i o n . In o v a r i e s a moderate ly p o s i t i v e r e a c t i o n

i s present in the c e l l s o f qerminal as w e l l as growth

zones which are a c t i v e l y engaged in m u l t i o l i c a t i o n .

( b ) A l ka l i n e phosnhatase

The a l k a l i n e phosphatase was found to be absent

in C. haukingi adult uorm.

F i g . 34. Photomicr-jqraph of T .S . o f adul t '3houing

ac id phosphatase a c t i v i t y in e p i t h e l i a l

c e l l s , qerminal and qrouth zones of

ovary and i n t e s t i n e . X 400.

F i q . 35. Photomicroqranh af T .' 3 . adul t shauing

ac id phosc.iT' 'j d J a c t i v i t v in mlunnar

Cells of rh;c(?r!taculun semin is , X 400.

£ n

139

•Z-ylB 'AO

'j'S-

•UAdS-J

T i g . 35. Photomicnqraph of T . 3 . of adul t showing

ac id phosphatase a c t i v i t y in uterus. X 400,

T i g . 37. Photomicnqraph pf T . 3 . of adul t shouing

ac id phosphatase a c t i v i t y in vag ina

u t e r ina , X 400.

140

ut.

F iq . 38. Photomicroq]3Dh o f s e c t i o n o f adu l t

shouing ac id phngphatase a c t i v i t y in

vag ina ve ra ( o b l i q u e S R c t i o n ) . X 400.

F i g . 39. PhotomicroL.raDh of T . S . of adu l t shouing

ac id phosph^tese a c t i v i t y on ly in

i n t e s t i n e j f male. X 400.

• -le 141

•••fe.

--J.

r Ti

1 int.^

k

T i g s . 40 41. Phot Tpi rrograohs Df T . 3 . of adul t

shouin^^ a r id phnsonatase a c t i v i t y

only in I n t e s t i n e of male. X 400.

( c ) Adenosine t r i p h o s p h a t a s e

R e s u l t s

The c u t i r i B , s u b - c u t i c l e and musculature shou a

modera te l y p o s i t i v e r e a c t i o n f o r t h i s enzyme. The

oesophagus shous a f e e b l e r e a c t i o n u h i l e the i n t e s t i n e

i s s t r o n g l y p o s i t i v e f o r t h i s enzyme.

In the f ema le r e p r o d u c t i v e o rgans , the qprminal

as u e l l as growth zones of the ovary show a p o s i t i v e

r e a c t i o n . The g l andu la r u a l l s of uterus and vag ina

shou a s t r o n g l y p o s i t i v e r p a c t i o n .

The enzyiTG a c t i v i t y i s found to be absent in

male r e p r o d u c t i v e o r g a n 3 ( F i g s . 4 2 - 4 5 ) .

D i s c u s s i o n .

Adenosine t r i phospha tas e occurs in areas uhere

c e l l s are i n vo lued u i t h o r in abso rp t i on or s e c r r t i o n or

t r a n s p o r t of m a t e r i a l o r a c t i v e t u 1 t i p l i c a t i o n .

In C. haukin'^i the muscu l=iturs shows a p o s i t i v e

r e a c t i o n as t h i s enzyme i s s y n t h e s i s e d in m i tochondr ia

o f muscle t i s s u e as demonstrated by Povyake l (1958)

in p i g A s c a r i s .

The i n t e s t i n e shows a p o s i t i v e r e a c t i o n as i t i s

i n v o l v e d in abso rp t i on and s e c r e t i o n as Ru i t onberg

(1972) has found a s t r ong a c t i v i t y in brush borders of

i n t e s t i n e of Anisakis sn. The nvarian c e l l s shou a

pos i t iwe r eac t i on ag the c e l l s are a c t i v e l y inv/olued

in d i v i s i o n in germinal as i i e l l as qrouth zones of

ovary in C. haukingi . Thn LIBIIS of uterus and vagina

are p o s i t i v e fo r th i s enzyme as some s ec r e t o r y processes

are going on in these s t ruc tures .

144

F ie . 46. Photomi cr 3qrapb of T . i . of adult snau 'ing

C 3 r b o x y l e s t e r a s e a c t i t / i t y i n g e r m i n a l

z o n e o r o v a r y a n d i n t e s t i n e . X 4 0 0 .

F i r . 47. Ph^ton icmor ' ' h ,f adult Jinuing

r ^ i b o x y l c - , t ' rc"3<= a c t i v i t y i n n r D U t h

zone Qp ovary. X /i-jT.

T i g . 42. Pho to r " i cn graph of L .S . of adult shouing

adenosine t r iphosohatase a c t i v i t y in

qrouth zone of ovary and g landular part

o f oDSophanus. X 4GO.

i g . 43. Phot ornicro'-^r'p'^ of L .S. of adult shouing

adenosine t r i p m s o h a t a s e a c t i v i t y in

germinal z nne of lUr-ry aod rectum. X 4Q0.

145

i

F i g . 44. Photomi c n a r a p h of L. 3 . nF adult shouing

adenosine t r inhogphatase a c t i i / i t y in

wa l l s o f uaqina. X 4.00.

T i g . 45. Photominronrarjh of L .3 . of adul t shouing

a d G m s i n e t r i o h o s p h a t a g e a c t i v i t y i n

c u t i c l e , m u s c u l a t u r e a n d v/uJva. TOO,

146

-vcjg.

"T'J

(d ) Carboxyl nstorase

R e s u l t s :

The body u a l l shows a f e e b l y p o s i t i v e r e a c t i o n .

The l a t e r a l e x c r e t o r y channels shnu s t r o n g l y p o s i t i v e

re a c t i o n .

Regarding the a l imentary cana l , only the i n t e s t i n e

shows a p o s i t i v e r e a c t i o n .

In the female r eproduc t i v e system, the germinal

zone and e p i t h e l i a l c e l l s c o v e r ing the o v a r i e s , shou a

p o s i t i v e r e a c t i o n . The wa l l s of o v i d u c t , the luminal

ep i the l ium of uterus and inner g landular l i n i n g of the

vag ina show s t r o n g l y o o s i t i v e r e a c t i o n .

In the male r eproduc t i v e system, l i n i n g of

e p i t h e l i a l c e l l s , c ove r ing the t e s t i s , p s o e c i a l l y o f

germinal zone are h igh ly p o s i t i v e f o r na rboxy l e s t e rase

a c t i v i t y . The e p i t h e l i a l c e l l s of seminal v e s i c l e a l so

shou s t r o n g l y p o s i t i v e re a c t i o n / F i g s . 46 -49 ) .

D i s cuss i on :

Workers l i k e Boqitsh (1956) and Halton (1967)

fouhd c a r b o x y l i c e s t e rase in tegumental r eg ions and

suggested that th i s enzyme takes part in nu t r i en t

t r anspo r t through tegumental r e g i o n s . They found the

e s t e rase to be nresent in oesoohagus and i n t e s t i n e and

148

presumed that t h i s enzyme takes part in e x t r a c o r p o r e a l

d i g e s t i o n . The presence oF th i s enzyme in teoumentary

and d i g e s t i v e system has been demonstrated by Bogi tsh

(1 966) •

In C. haukinpi , the i n t e s t i n e shouing p o s i t i v e

r e a c t i o n goes to prov/e that i t takes some part in

d i g e s t i o n . A p o s i t i v e r e a c t i on shoun by e p i t h e l i a l

l i n i n g of male and female r ep roduc t i v e system a f f i r m s

the view that these l i n i n g s take part in nut r i en t

t r a n s p o r t .

A s t r ong l y p o s i t i v e r e a c t i o n e x h i b i t e d by l a t e r a l

e x c r e t o r y channels Further support the v i eu that t h i s

enzyme takes part in the t ranspor t of the e x c r e t o r y

f l u i d .

. - 149

. . M ov.germ.z.

\ •

F iq . 48. PhotnmicrDQraph of T .5 . of adult shouiing

carboxy l es t e rase a c t i v i t y in u t e r i i . X 400,

^ iq . 49. Photornicn "^rcrj'i )f T .3 . nf adult shouinq

carboxy l 'ist-iT-iar-- act lu ity >.n e p i t h e l i a l

c e l l s of t p s t i s . X 400.



( b ) A l ka l i n e phosph.'tase

( c ) Adinosine t r iphosphatase

( d ) Carboxyl e s t e rase



R e s u l t s :

The d i s t r i b u t i o n and i n t e n s i t y o f ac id phosphatase

a c t i v i t y in the m i c r o f i J a r i a of £. haukingi i s presented

in Tab le 6. Smears incubated in subs t ra te f r e e medium

showed no r eac t i on product in m i c r o f i l a r i a .

Strong ac id phosphatase act iv/ i ty uas seen in

''lungebi I de , e x c r e t o r y v e s i c l e , G c e l l and anal v e s i c l e .

Comparat ive ly f e e b l e a c t i v i t y uas observed in the f i r s t

f eu c e l l s of the nuclear coluTin, sub - cu t i cu l a r c e l l s

o f a n t e r i o r ha l f of the body, c e l l s and l a s t

tuo c e l l s of ^hp nuclear colunn, oresent near the t i p

"of the t a i l ( F i q s . & 51 ) ,

D iscuss ion •

The d i s t r i b u t i o n of the acid phosphatase a c t i v i t y

in m i c r o f i l a r i a i s so s n e c i f i c that i t o f f e r s a parameter

f o r d is t inqui s h i n o ths pp^nera, s ' e c i c s and even the

s t r a i n s o f f i l a r i a l unrms. Thus s e v e r a l i n v e s t i g a t o r s

d i s t i n g u i s h e d the s p e c i e s o f f i l a r i a l uorms by l o c a l i z i n g

the d i s t i n c t pa t t e rns of ac id phosohatase a c t i v i t y in

c i r c u l a t i n g rn i c ro f i l a r i a e . C h a l i f o u x and Hunt ( 1 9 7 1 ) ,

Balbo and Abate (1972) d i s t i n g u i s h e d the s p e c i e s o f

D i r o f i l a r i a from Dipeta lonema even in mixed i n f e c t i o n s .

R e d i n g t o n , Plontgomery, Gerv i s and Hockmeyer (1 975 )

d i s t i n g u i s h e d h i s t o c h e m i c a l l y the m i c r o f i l a r i a e o f

Brugia malay i from B. pahangi . Teruedou anri Huff ( 1 9 7 6 )

l o c a l i z e d t h i s enzyme in LJuchereria b a n c r o f t i . Hmar

(1977) con f i rmed the o b s e r v a t i o n s o f p r ev i ous workers

and l o c a l i z e d the enzyme a c t i v i t y in d e v e l o p i n g s t a g e s

in the v e c t o r o f a l l the four f i l a r i a l worms v i z . ,

Uuchere r i a b a n c r o f t i , Brugia m a l a y i , 8. pahangi and

D i r o f i l a r i a i m m i t i s .

Braun-f^unz inqer and Sputhgate (1 977) uent s t i l l

f u r t h e r by di f f e r f? n t i at ing the f our d i f f e r e n t s t r a i n s

o f Onchocerca v o l v u l u s by l o c a l i z i n g aciri phosphatase

in the micro f i l a r i ae (TableT)•

The d i f f e r e n t areas p o s i t i v e f o r ac id phosphatase

i n d i c a t e that t h i s enzyme a c t i v i t y i s p resent on l y at

m e t a b o l i c a l l y a c t i v e r e g i o n s . I t i s supposed t h a t ac id

phosphatase a c t i v i t y i s a s s o c i a t e d u i t h c e r t a i n impor tant

f u n c t i o n a l p rocesses such as a b s o r p t i v e , p h a g o c y t i c ,

e x c r e t o r y or s e c r a t o r y a c t i v i t i e s o f the l i v i n g c e l l s .

153

The m i c r o f i l a r i a of C. haukinqi shous s t rong

a c t i v i t y at the a n t e r i o r end correspondinq to PlungebiJrie

or amphi is , probably due to i t s abso rp t i v e f unc t i on .

S i m i l a r l y the s t rong r e a c t i o n in the e x c r e t o r y and anal

v e s i c l e s i n d i c a t e s the involvement of the enzyme in

e x c r e t o r y or s e c r e t o r y processes . The s i g n i f i c a n c e of

enzyme a c t i v i t y in the C^, R^ and R^ c e l l s , and the

f i r s t feu and l a s t tuo c e l l s of the nuclear column i s

not c l e a r . I t appears , however, that the enzyme i s

i n v o l v e d in c e l l p r o l i f e r a t i o n , s y n t h e s i s , s e c r e t o r y

and absorp t i ve f u n c t i o n s .

The i n t e n s i t y of a c t i v i t y of t h i s enzyme at

Plungebilde i s very s t rong and almost s i m i l a r to that o f

D i r o f i l a r i a immi t i s . S i m i l a r l y the a c t i v i t y in the

e x c r e t o r y v e s i c l e resembles that of B. ma lay i ,

U. b a n c r o f t i and immit is but not B. pahangi in uhich

compara t i ve l y lou a c t i v i t y has been recorded . Here

Innenkorper showed a strong r eac t i on corresponding to

tha t of U. b a n c r o f t i .

Anal v e s i c l e shous s trong a c t i v i t y f o r t h i s enzyme.

This s t ruc tu re shous equa l l y s t rong a c t i v i t y in a l l the

desc r i bed s p e c i e s . Uar iab le i n t e n s i t y of enzyme a c t i v i t y

uas shoun by Schuanzgehi lde and sub - cu t i cu l a r CGlis in

the d i f f e r e n t s p e c i c s .

154

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F i g . 50, Photomicro qraph of lonn form o^ m i c n f i l a r i a

shouino ac id phosphatase a c t i v i t y . X 900.

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micri '-MlcMiP nhnuing acid phosnhatase

a c t i v i t y . X 400.

157

t

158

( b ) A lka l ine phosphatase

A l ka l i n e phosphatase uas found to be absent in

m i c r o f i l a r i a of C. haukingi .

159


Resu l ts "

adenosine t r i ph isphatase i s in t ense l y present

in the cepha l i c end, cxc r e t o r y v e s i c l e and G c e l l .

The sheath also shows a f e e b l y p o s i t i v e r e a c t i o n ( F i g . 52)

D i s cuss i on :

Adenosine tr iphosphatase is assoc ia ted u i th

energy metabolism, pembrane t r anspo r t , muscle t i s sue

format ion and absorpt i ve func t i ons . The strong ATPase

a c t i v i t y , e xh ib i t ed by the cepha l i c end, exc re to ry

v e s i c l e and G c e l l indicates the functional a c t i v i t y

and energy rretabolisr" in thesc^ s t ruc tu res .

The cophai i c end takes part in absorpt ion or

membrane t ranspor t of food m a t e r i a l ; the e x c r e t o r y

v c s i c l o shous a strnnq r eac t i on f o r t h i s enzymo, as i t

i s assoc iatpd u i f - tbt metabo l ic processes . G c e l l

shous a c t i v i t y , as i t i s r i ch in chnmat in mater ia ] and

i s f i r s t to d i v i de during development.

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JO ujjoJ 5uo-[ JO L|deJboaoTUJo::^oq(j • 'OT j

160

'.v. 1

161

( d ) Carboxyl o s t c rase ( n o n - s p e c i f i c )

Re su I t s

An intense n n n - s o e c i f i c c i r b o x y l e s t e r a se

a c t i v i t y uas 'observed in the c enha l i c r eg i on of

m i c r o f i l a r i a e from a n t e r i o r t i p to the nerve r i n g , in

the e x c r e t o r y v e s i c l e , anal v e s i c l e and one or tuo

c e l l s near the t i p of t a i l . A f eu sub - cu t i cu l a r ce U s

a l s o shoued the carboxy l e s t e r a s e a c t i v i t y ( F i q s . 53 & 54 ) .

D i s cuss i on :

Carboxy l l c estrar hydro lases ( c a rboxy l e s t e r a s e s )

are a l a r g e group of n o n - s p e c i f i c hydro lases a c t ing at

pH optima betuecn 5.0-9,N on simple shor t - cha ined f a t t y

ac id e s t e r s . They r,rn both h y d r o l y t i c and s y n t h e t i c

in a c t i o n . Houiever, ib i s t r u e , as layers (1960) has

po inted out , that the "metabolic func t i ons of the

e s t e r a s e s are not comple te ly knoun.

Tho strong e s t e rase a c t i v i t y in the a n t e r i o r

par t o f the m i c r o f i l a r i a i nd i c a t e s a pos s i b l e f unc t i on

o f tho enzyme in absorpt ion and r e syn thes i s of f a t t y

ac id e s t e r s . The enzyme a c t i v i t y in the nerve r ing

r e g i on might represent a cho l i nos t e r a s e a c t i v i t y s ince

oC-naphthy l a c e t a t e i s a lso hydro lysed by c h o l i n e s t e r a s e s .

Houever, the p o s s i b i l i t y that the e s t e r a s e s are i n vo l v ed

in the synthes is of l i p i d s , c h a r a c t e r i s t i c of nervous

system cannot bo ove r l ooked .

S i m i l a r l y the i^cl l knoun a s s o c i a t i o n of e s t e r a s e

a c t i v i t y ui th c x c r o t o r y func t i on in many animals , o f f e r s

an exp lana t i on f o r tho intense enzyme a c t i v i t y seen in

the e x c r e t o r y and anal v e s i c l e s of m i c r o f i l a r i a .

In the c e l l s o f the p o s t e r i o r end of the

m i c r o f i l a r i a , e s t e r a s e s might be i n vo l v ed in c e l l

p r o l i f e r a t i o n or s y n t h e t i c f u n c t i o n s .

162

F i g . 53. Photomicroaraoh of long form o f

m i c r o f i l a r i a ahouinq narboxy l e s t e rase

a c t i v i t y . X 900.

F i g . 54. Photomicroqrar'h of short form of

m i c r o f i l a r i a shouinq carhoxy l e s t e rase

act iv/ i ty . X QOO.

164

( s ) A r y l 'Sulphatase

R e s u l t s :

A d i f f u s n d r ea c t i on a l l over the body has been

obseruedJ^Figs. 55 & 56) .

D i scuss ion ;

The e n t i r e body of the m i c r o f i l a r i a , ra ther the

nuclear column, showed r e a c t i o n f o r n ry l sulphatase

uhereas tho Innenkorpor reg ion gave a nega t i ve r e a c t i o n

in long forms and in short forris a d i f f u s e d r e a c t i o n

i s o resent along the e n t i r e l ength of the body inc lud ing

Innenkorper .

F i g . 55. Photomicrograph of long form of

m i c r o f i l a r i a showing a r y l sulohataao

a c t i v i t y . X 900.

F i g . 56. Photomicroqrrnh of short form of

m i c r o f i l a r i a shouiino n ry l sulphatase

a c t i v i t y . X TOO.

165

-A-

166

4. In v i t r o c u l t i v a t i o n

A l l the ijn v i t r o exper iments uere c a r r i e d out

at 22 C and at the pH 7.5 ad justed with the he lp of

0 .4^ NaHCOj or T r i s b u f f e r and t e s t e d with u n i v e r s a l

i n d i c a t o r .

Experiment No." 1

Ty rode ' s s o l u t i o n (Tyrode , 1910)

R inger-Locke (Locke , 1901)

Hank's BSS (Hanks and Wal lace , 1949)

and E a r l e ' s s a l i n e ( E a r l e , 1943) uere taken

and supplemented with 20 or 50^ of i n a c t i v a t e d bovine

SB r urn.

The m i c r o f i l a r i a e surv i v ed f o r 6-9 days, both in

Tyrode and Rinqpr-Locke uhen supplemented u i th 20/S bovine

serum. The s u r v i v a l t ime became 11-13 days u i th 50/S

serum supplement.

The s u r v i v a l time did not improve much in Hank's

BSS and E a r l e ' s s a l i n e . Uith 20w serum supplement the

s u r v i v a l time uas 5-10 days and u i th 50^' the s u r v i v a l time

became 10-12 days.

No grouth or development could be observed .

Usual ly the m i c r o f i l a r i a e remained ve ry a c t i v e f o r

4-5 days and then they s t a r t e d becoming s lugg i sh and

dy ing g radua l l y .

167

Experiment No. 2

Chemical ly d e f i n ed medio, NCTC 109 (Evans, Bryant ,

F io ramont i , f^cQuilkin, S a n f o r - , U e s t F a l l and Ea r l e , 1956;

McQuilkin, Evans and Ea r l e , 1957).

Eaq l e ' s MEM (Eag l e , 1959)

CnRL - 1066 ( Pa rke r , Cas tor , NcCul lock, 1957)

Grace 's Gi A (Grace , I95f0 uere used supplsmentinf j

^ u i th 20% or 50% bovine serum,

( M i c r o f i l a r i a e surv i v ed f o r 10-12 days in NCTC 109

and in medium 199 u i th 20% serum supplement s l i g h t

inc rease in length (6 j j ) occurred .

The s u r v i v a l time uas reduced to 5-5 days uhen

serum supplement u ' s increased to 50%, I t uas noted

that there uas a gradual decror'se in the pH of the

medium uhich became a c i d i c ( t ) -4 ) a f t e r 5 nays. I t i s ,

t h e r e f o r e , conc luder that e i t h e r the m i c r o f i l a r i a e could

not stand the a c i d i c pH or the concent ra t i on of the

media, uhich may be too r i c h .

The m i c r o f i l a r i a e surv i ved f o r 14 days in ClRL-IOeS,

They remained very a c t i v e in Grace ' s medium uhen

supplemented u i th 20% bovine serum and surv i ved f o r

20-24 days. No growth or development uas observed ,

50% serum supplement uas not used.

168

Experiment No. 3

E a r l e ' s s a l i n e , R inger-Locke and Hank's B53

supplemented with lacta lbumin 5%, yeast e x t r a c t 2%.

In a d d i t i o n , 20/ serum of uh i te ra t or guinea p ig or

bov ine or f o u l were used.

In Ear l a ' s medium, having the above mentioner;

percentage of lac ta lbumin and y e a s t , uhen supplemented

u i th 20^ uhi te ra t serum, the m i c r o f i l a r i a e surv i v ed

f o r 12-16 days; u i th 20'^ guinea p i g serum the s u r v i v a l

t ime became 15-17 days; u i th 20^ bovine scrum, the

m i c r o f i l a r i a e surv i v ed f o r 8-10 days and u i th 2 0>o f o u l

s'erum f o r lR-20 days.

In Rinnor-Locke and Hank's BSS the s u r v i v a l time

did not improve. The range of s u r v i v a l time i s almost

the s 'me.

Experiment No. 4

This cxneriment u- s done by using E a g l e ' s nE* ,

nedium 199, NCTC 109, C' ^IRL-IOee -" nd Grace 's Gi'lA. The

m i c r o f i l a r i a e kept comparat i ve ly more a c t i v e , the

s u r v i v a l time uas increased and sometimes there uas

l i t t l e inc rease in s i z e .

liihen E a g l e ' s NEn, having lacta lbumin (5%) and

yenst e x t r a c t (25^), supplemented u i th 2 0^ uh i te ra t

serum, uas used f o r c u l t i v a t i n g m i c r o f i l a r i a e , they

su rv i v ed f o r 7-10 days but no grouth or development uas

169

obse r v ed ; when guinea p i g sarum was supplemented,

the m i c r o f i l a r i a e surv i v ed f o r 10-11 days, they kept

q u i t e a c t i v e u i th 20^ bovine serum and surv i ved only

f o r 6-8 dcys . Uith f o u l serum supplement the

s u r v i v a l time inc reased to a maximum pe r i od of 20 days.

T h e r e a f t e r the m i c r o f i l a r i a e s t a r t e d becoming s lugg i sh

and dying g radua l l y .

The r e s u l t s in medium 199 and NCTC 109 were

almost s i m i l a r . IMCTC 109 u i th 2% yeast e x t r a c t and 5%

l a c ta lbumin , uhen supplemented with 20% uh i te ra t

serum, the m i c r o f i l a r i a e surv i v ed f o r 12-15 days and

in Medium 199 thoy surv i ved f o r 12-16 days. In both,

s l i g h t increase in length (4 yu) uas observed ; uhen

NCTC 109 and medium 199 were supplemented with 20';

guinea p ig serum, the s u r v i v a l t ime became 17-19 days

in both the media. n i c r o f i l a r i a e kept very a c t i v e and

perhaps due to the u r i g q l i n g of m i c r o f i l a r i a e , the

sheath at the a n t e r i o r end got broken. An increase of

6 yu in length was observed ; uhen supplemented u i th 20%

bov ine serum the m i c r o f i l a r i a e surv i v ed f o r 10-12 days

in NCTC 109 and 8-14 days in medium 199; uhen

supplemented with 20% f o u l serum, the m i c r o f i l a r i a e

su r v i v ed f o r 15-20 days in NCTC 109 and f o r 17-20 days

i n medium 199.

Yeast e x t r a c t (2%) and lacta lbumin (5%) uere

added to CnRL-1066 and Grace 's medium a l so and then 20/

o f d i f f e r e n t sera supplements uere g iven f o r c u l t i v a t i o n

o f m i c r o f i l a r i a e .

In CflRL-1066 u i th 20% uh i t e ra t seru^r), the

m i c r o f i l a r i a e suruiued Tor 14 days; u i th 20% guinea p i g

serum the m i c r o f i l a r i a e suruiued f o r 18-22 days,

breaking of sheath and inc rease in l ength (6 /u) uas J

observed ; with 20% bovine serum they surv i v ed f o r

12-13 days; with 2 0% f o u l serum the m i c r o f i l a r i a e

su r v i v ed f o r 20-24 days.

The Grgce ' s GMA, gave the best r e s u l t s . When

supplemented u i th 20% white ra t serum, the m i c r o f i l a r i a e

kept ve ry a c t i v e , the shenth usua l l y got broken. They

su r v i v ed f o r 14-18 days; with 20% guinea p i g serum

supplement, the m i c r o f i l a r i a e s u r v i v e d f o r 20-22 days;

w i th 20% bovii.e serum supplement, the- m i c r o f i l a r i a e

surv i v ed f o r 12-15 days and u i th 20% f o u l serum

suoplement the m i c r o f i l a r i a e surv i v ed f o r 20-28 days.

Experiment No. 5

In th i s exper iment the m i c r o f i l a r i o e uere

c u l t i v p t e d in Grace ' s G( A and CnRL-1066 supplemented

u i t h 20% crou serum, crou b lood ce l i s ( separa ted by

adding c i t r a t e , d i l u t i n g and c e n t r i f u g i n g ) , minute

f ragments of Aedes l a r v a l gut , 0.5% Aedes (pupa l )

haemolymph, 1.0% g lucose and 0.5% CEE.

The s u r v i v a l t ime inc reased to 25-30 days in

CnRL-1066 and 43-45 days in Grace 's GI A. In Grace ' s

GNA, the m i c r o f i l a r i a e surv i ved f o r the l onges t t ime .

171

most of them showed broken sheath, vet there uas no

t rue ecr iys is , a feu became shortened and th ickened with

broken sheath s t i l l i n t a c t . i o t rue deunlopmental

s tages could be obseruod.

Experiment No. 6

nonolayGrs i f white ra t embryo, chick embryo,

h ea r t , kidney and l i v e r (uh i te rat p r e n a t a l ) uere prepared

and o v e r l a i d u i th Hank's P3S, lacta lbumin 5%, yeast

e x t r a c t 2/ and uhi te rat serum IT"^.. The m i c r o f i l a r i a e

su r v i v ed f o r 5-7 days and then '^ost of them nenetra ted

under the monolayer and d i ed . c3ome of them uero found

l y i n g dead under the monolayer even a f t e r 24.0 hours.

d i s c u s s i o n :

The m i c r o f i l a r i a e of Chand le re l l a haukingi

su r v i v ed f o r about 10-12 days in NCTC 109 and medium 199

u i t h 20% scrum supplf^ment, uh i l e the m i c r o f i l a r i a e of

M r o f i l a r i a immit is ( E a r l , 1959) , surv i ved f o r 4 days

in medium 199 a l one , f o r 43 days u i th 10'/. scrum

supplement and f o r 61 days u i th serum supnlomont.

In the present case , s u r v i v a l time of C. haukingi

decreased to S-6 Hays only uhen the serum supplement

uas increased to 50%.

The s u r v i v a l time of C. haukinqi m i c r o f i l a r i a e

i s compara t i ve l y longer than that of D. immit is

m i c r o f i l a r i a e ' ' jauiytr and U'^ instp in, 1961) uhen cul t iv/ated

in V/itro in R inqer and Locke ' s s o l u t i o n u i t h d i f f e r e n t

supplemonts , Sauy j r and U e i n s t e i n ( 1 9 5 1 ) had used

Krebs -K inge r phosphate i/ith g lucoso and ^ .Sf- sodium

c a s e i n a t e and C. haukingi m i c r o f i l a r i a e uere c u l t u r e d

i n Tyrodo and Hinq^r L i c k e ' s s o l u t i o n wi th 20% bov ine

se rum.

The m i c r o f i l a r i a e of D, immit i s surv/iued f o r

7 days (Sauyer and U e i n s t e i n , 1161) in NCTC 109 and the

m i c r o f i l a r i a e o f C. haukingi s u r v i v e d f o r 10-12 days

i n t h i s medium.

The s u r v i v a l t ime o f m i c r o f i l a r i a e of C. hauking i

in GPIA wi th 20) sprum supplement remained only f o r

20-24 days but the m i c r o f i l a r i a e o f naracanema formosana

(Uood and S u i t o r , 1966) dev(^ loped to i n f e c t i v e s t age

in GnA supplementpd u i th D.S^ i n s e c t haemolymph and 1

f o e t a l bov ine serum.

Bcjsides a l l t h e s e , a number of o ther rombinai-ions ,

c h c m i c a l l y de f in t d m fd i a , ^upnlemented u i th v a r i o u s

n u t r i e n t s and TC mono layers , o v e r l a i d u i t h growth

media , having Hank's 653, l a c t a l b u n i n , yr.ast e x t r a c t

and serum, have been used f o r c u l t i v a t i n n v i t r n ,

the m i c r o f i l a r i a e nf C. haukinni . Th j r e s u l t s

uerp ob ta ined uhen c u l t i v a t ' - d in G!'1A supolemented

173

ui th 20f> ' r r o u S f ^ r u r r , n r o u b l o T d C f j l l s , minute fragments

of Aode3 l a r v a l qut and 0.5/^ Aedes ^pupal) haumolymph,

1.0% g lucose and 0.5'/^ CEE. In such a medium the suruiual

time increased to days.

174

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ABPREUIATI3NS

223

a. p.

b. rn.

c . s .

c a p . c .

c i r.mus.

c o n . t .

c o n . t . s u p .

cu.

cu, s .

cu. th .

d . e j .

d o r s . c h .

e . p.

f . c . n. CO 1.

f i b . 1 .

f i b . l a m .

f i b . z .

f i b r . 1 .

S

g l . o e s .

h.

i n t .

i n t . l u .

1 . c . n . c o l .

anal pore

basal membrane

c epha l i c space

cap c e l l

r i r c u l a r muscles

connectivye t i s sue

connec t i ve t i s sue support

cu t i c le

c u t i c u l a r s t r i a t i o n s

c u t i c u l a r th i cken ings

ductus e j a c u l a t o r i u s

f lorsa l chord

e x c r e t o r y pore

f i r s t c e l l of nuclear column

f i b r i l l a r l a y e r

f i b r i l l a r l ame l la

f i b r i l l a r zone

f i b r o u s l aye r

G c e l l

q landular oesophagus

hook

i n t e s t i n e

i n t e s t i n a l lumen

l a s t c e l l o f nuclear column

E 2 4

1. gn.

mat. 1.

m f .

mu3.oes.

muse,

n. r .

nu.

0 .

o b i . mus.

oogn.

o\j.

a\j. d.

o\y. germ, z .

o\/. gr . 2 .

p. t .

r .sem.

re c t .

re c t . c .

s .

sem.ves.

som.int.mus,

som.muse,

som.oes.mus .

sp. s.

s p l . z .

l a t e r a l gangl ion

matrix layer

m i c r o f i l a r i a

muscular oesophagus

musculature

nerve r ing

nucle us

o r a l r ing

ob l i gue muscles

oogonia

ovary

ov iduct

ouary germinal zone

ouarv qrouth zone

pharyngeal thread

receptaculum seminis

re ctum

r e c t a l c e l l

spi ne

seminal v e s i c l e

somato i n t e s t i n a l muscles

somatic musculature

somato oesoohageal muscles

sp i cu la r sheath

splanchnic zone

225

spmt gon. snRrmatoQonia

sp r . sf.ierm

s p r z . snermatozoa

t . t e s t i s

t . germ, z . t e s t i s germ zone

t . g r . z . testis growth zone

ut . uterus

V ag. u aoina

wag. ut. vagina uter ina

uag. v/er. V/ agina uera

v a s . d e f . \jas de f e r ens

SUI NARY

The thes i s embodies the r e s u l t s of the s tud i e s

on Chand l e r e l l a hawkingi , a f i l a r i a l p a r a s i t e of Ind ian

jung l e crouj, Corvus macrorhynchos ( U a g l e r ) . Only four

a spec t s , morphology, h i s t o l o g y , h i s t ochemis t r y aad

in V i t r o cu l ture have been taken up.

The morpho log i ca l s tud i es inc lude a re d e s c r i p t i o n

of adul t uorm u i th an add i t i on of a feu minor d e t a i l s

in the adu l t . The s t ruc tures descr ibed in m i c r o f i l a r i a

are the nuclear s t r u c t u r e s , c epha l i c s t r u c t u r e s ,

pharyngea l thread , Innenkorper and a l so occurrence of

tuo forms o f m i c r o f i l a r i a e in blood of crou.

The nuclear s t ruc tures hav/e been s tudied u i th

s p e c i a l r e f e r ence to nuclear landmarks which are of

g r ea t taxonomic va luo .

Sometimes the nuclear s t ruc tu r e s do not s u f f i c e

f o r i d e n t i f i c a t i o n of genera and s o e c i e s , the c e p h a l i c

s t r u c t u r e s , pharyngeal thread and Innenkorper have a l so

been s tud i ed .

I t has a l so been proved that the tuo forms of

m i c r o f i l a r i a e , the long and the sho r t , present in the

blood of crou be lonc to the same s o e c i e s , C. hauking i .

265

The stereosnan s tud ies of the c u t i c u l a r s t ruc tu r e s

of the adult and the m i c r o f i l a r i a haue been done as these

are of g rea t taxonomic importance in c l a s s i f i c a t i o n of

uorms.

The h i s t o l o g i c a l s tud i es inc lude the study of

h i s t o l o g i c a l f e a t u r e s of the body u a l l , musculature,

a l imentary canal and r eproduc t i v e organs as some of these

s t ruc tu r - prov ide a taxonomic t o o l at var ious l e v e l s ,

iiq h i s tochemica l -^^.tudies, four enzymes, v i z ;


t r i phospha tase and ca rboxy l e s t e r a s e have been l o c a l i z e d

as these enzymes are a t t r i b u t e d to d i f f e r e n t func t i ons

and t h e i r d i s t r i b u t i o n i s very s p e c i f i c . Thus the

l o c a l i z a t i o n of these enzymes hr-slos in determining the

var ious func t i ons assigned to d i f f e r e n t organs and par ts

of the body of adult uorm.

In m i c r o f i l a r i a , f i v e enzyn-93, ac id phosphatase,

a l k a l i n e phosphatase, adenosine t r i phosoha tase ,

c a r b o x y l e s t e r a s e and a r y l su lphatase , have been s tud i ed .

The d i s t r i b u t i o n of these enzymes i s so s p e c i f i c that

t h e i r pa t t e rns help in i d e n t i f i c a t i o n of d i f f e r e n t

genera and s p e c i e s .

228

In the prnsent u.'ork only n i c n F i l a r i a e of

C. haukinqi have been cult iv/ated in v i t r o , in v i eu to

study t h o i r dcvelopment outs ide thn body of tho hos t .

These s tud ies are important even f o r t f j s t i n g the drugs

on p a r a s i t e alone and a l so f o r c o l l e c t i o n of E5 products

uhich may serve as f u n c t i o n a l ant igens aga ins t

f i l a r i a s i s .

STUDIES ON CHANDLERELL HAWKiNCfA , A FILARIA PARASITL …ir.amu.ac.in/1891/1/T 5322.pdf · ABSTRACT...

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