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 IN . /nomRY

SHAHNAZ BANO

SECTION OF DEPARTMENT OF ZOOLOGY ALIGARH MUSLIM UNIVERSITY ALIGARH

January, 1981 ABSTRACT

The thesis embodies the results of the studies on Chandlerella haukingi, a filarial parasite of Indian jungle crou, Corvus macrorhynchos (Uagler). Only four aspects, morphology, histology, histochemistry and in ^itro culture have been taken up.

The morphological studies include a re description of adult worm uith an addition of a feu minor details in the adult. The structures described in are the nuclear structures, cephalic structures, pharyngeal thread, Innenkorper and also occurrence of tuo forms of microfilariae in blood of crou.

The nuclear structures naue been studied uith special reference to nuclear landmarks which are of great taxonomic vyalu^.

Sometimes the nuclear structures do not suffice for identification of genera and soecies, the cephalic structures, pharyngeal thread and Innenkorper have also been studied.

It has also been proved that the tuo forms of microfilariae, the long and the short, present in the blood of crou belono to the same species, C. haukingi. The stereoscan studies of the cuticular structures of the adult and the microfilaria have heen done as these are of great taxonomic importance in classification of uorms.

The histological studies include the study of histological features of the body uall, musculature, alimentary canal and reproductiue organs as some of these structures provide a taxonomic tool at various levels.

Among histochemical •^^tudies, four enzymes, viz; acid phosphatase, alkaline phosphatase, adenosine triphosphatase and carboxylesterase have been localized as these enzymes are attributed to different functions and their distribution is very specific. Thus the localization of these enzymes helos in determining the various functions assigned to different organs and parts of the body of adult uorm.

In microfilaria, five enzyres, acid phosphatase, alkaline phosphatase, adenosine triphosphatase, carboxy lesterase and aryl sulphatase, have betin studied.

The distribution of these enzymes is so specific that their patterns help in identification nf different genera and species. In the present uork only microfilariae of

C. haukingi have been CLiltivated in vitro, in vieu to study their development outsidf-i the body of the .

These studies are important even for testing the drugs on parasite alone and also for collection of ES products uhich may servs as functional against . 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 llipri) Muslim ALIGARH. M. P. 202001 PARASITOLOGY RESEARCH LABORATORY ^Enibersiitp DEPARTMENT OF ZOOLOGY

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

This is to certify that the thesis entitled, "Studies on Chandlerella hamkingi« a filarial parasite of crow", which is beirrg submitted by Tslrs. Shahniaz Banoi, embadies original urork done by the candidate herself. The entire toark was carried out under my stipervisipn. betuieenB 1976-1980 and that I alloui her ta submit the same in fulfilment 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 sincere thanks to Prof.

Ather H. Siddiqi to uhom I am indebted for his

supervision, guidance, invaluable suggestions,

constructive criticism and encouragement throughout

this uork.

I am thankful to Prof. S.i^. Alam for providing

me necessary facilities in the Department of Zoology.

I am grateful to Dr. Nitya Nand, Director,

Central Drug Research Institute (CDRl), Lucknou, and to

Dr. A.B. Sen, Head, Parasitology Division, for the use of research facilities in the Central Drug Research

Institute.

The help given by Dr. Ran Govind, Scientist,

Division of Parasitology, CDRI , is gratefully acknouledged,

for he permitted me to use all the facilities available in his laboratory, especially for the use of his culture

room and for providing all chemically defined culture media and for giving valuable suggestions.

( SHAHIMAZ BAIMO ) N T E N T S

Page No.

I . INTRODUCTINN 1 - 7

II. HISTORICAL REUIEU 8 -49

III. STATEI^ENT GF PRGBLEN 50 -53

L\J. MATERIALS AND HETHOOS 54 -77

\J, RESULTS AND DISCUSSION

1. Morphology (a) Light microscopy 78-104 (b) Stereoscan studies 105-110

2. Histolo gy ... 111-135

3. Histochemistry ... 136-165

4. In vitro cultivation 165-173

\LL, REFERENCES 174-222

\YIL. ABBREVIATIONS 223-225

Win. SUr^HARY 226-228

*** * INTRODUCTION

Among the uhich include a uery large number of worms belonging to different groups, the filariids are of great medical and veterinary importance.

They cause much human suffering in many parts of the uorld, especially in the developing countries. In early stages of infection the patient suffers from chills, fever, aches and general malaise. This, if neglected, results in of lymph glands (lymphadenitis) and lymph channels (lymphangitis), thereby starting allergic reactions ending in elephantiasis. The latter is characterised by enlargement of certain parts of the body due to the deposition of extracellular protein, fibrosis and necrosis of tissues, causing physical discomfort as uGll as disabilities resulting in much mental agony.

Not only that, these filarial worms cause tropical eosinophilia, if accidentally, man gets infected uith filariae of origin uhich cannot develop satisfactorily in man. These filarial worms may cause disturbances in eyes and central nervous system also.

The most common human filariids are Uuchereria bancrpfti and Bruq5 a malayi. U. bancrofti is prevalent in warm and humid countries like India, Africa, China,

Arabia, Nalaya, Formosa and West Indies; uhereas B, malayi is found in India, nalaya, Southeastern Asia and East

Indies etc. These are the worms commonly responsible for

lymphangitis, lymphadenitis and elephantiasis. The other

filariids infecting man are uolv/ulus,

Dipetalonema perstans, D. streptocerca, l^ansonella ozzardi,

Loa loa and spp.

Ivulus occurs in Plexico, Guatemala, Salvador,

Northuest , Africa and . It causes , resulting in deuelopment of fibrous nodules in the skin. Dipetalonema perstans is Found in Congo,

Uganda, South America and northern Argentina. D. streptocerca is found in Liest and Central Africa. No evident symptoms are produced except a persistent headache, drousyness, skin rash and eos inophi li a. Plansone 11a ozzardi is common in West Indies, Yucatan, Panama, South America and in northern Arqentia. The uorms are found in mesentries and visceral fat. No pathogenic symptoms are produced, infection results in an increase of blood eosinophils.

Loa loa is common in Uest and Central Africa. Adults live in sub-cutaneous tissue of man and make excursions from place to place under skin causing itching and creeping sensation. They shou a special preference for creeping in and about the eyes. It causes painless or itchy edematous suellings called "Calabar suellings" which appear suddenly, last a feu days and then disappear, to reappear later someuhere else. These are due to the

allergic reactions, to metabolic products of the uorms or

to proteins liberated from injured or expired uorms. flicrof-ilaria loa may cause fatal encephalitis, uhen they

penetrate into the brain and spinal cord.

Gne species of Dirofilaria, 0. conJuctivae has

been found to parasitise man in Europe, India, U.S.S.R.

and Thailand. It lives in cyst like tumors of eye, nose,

arm and mesentry.

The filarial parasites of domestic belong

to the species of Brugia, Dipetalonema, Onchocerca,

Setaria, Stephanofilaria, Parafilarja and Dirofilaria.

The species of Brugia parasitising cats and dogs

are, B, malayi in India (Orissa) , P'lalaya and Kenya coast

of Africa; B. pahanqi in Malaya, and B. patei in Pate

island and Kenya. They do not produce any pathogenic

symptoms. Only one species of Dipetalonema, D. reconditum

is a very common parasite of dogs in U.S.A. but it does

not produce any pathogenic symptoms.

The species of Onchocerca infecting domestic

animals are, £. qibsoni parasitic in .^ It injures

hides and carcasses by forming hard nodules, 0. indica

and 0. gutterpsa infect cattle and they also form skin nodules. 0. reticulata (O. cervicalis) inhabits the neck

ligament of horses in U.S.A. causing "poll ill" and fistulous withers, the microfilariae cause papular itching skin sores. 0. armillatus causes aneurysms in aorta of cattle in Africa,

The species of Setaria parasitic in domestic animals are, 5. equina in horses, 5. labiato-papillosa,

5. cervi and digitata in cattle in India. They do not cause any damage, but in non-specific hosts, they are carried even to the brain and there is a danger of invasion of central nervous system of sheep, goat and horses etc.

The 'epizootic cerebro-spinal nematodiasis' is found to be caused by immature S. cervi, besides this, lumbar paralysis or 'Kumri' has also been caused.

The species of Stephanofilaria parasitising cattle are S. assamensis causing humpsore in cattle in Assam,

Bengal, Bihar and Orissa, zaheeri causing ear-sore in

Hyderabad and stelesi causing skin and humpsore in cattle, goat and pig in India.

The species of Parafilaria infecting domestic animals are P. bpvicola in cattle and P. multipapillosa in horses in old uorld. The uorms pierce the skin to deposit embryos, causing "summer bleeding" from small

"Modules and injuring the hides.

The species of Dirofilaria infecting domestic animals are D. scaoiceps parasitic in rabbits, D. immitis and D. repens parasitic in dogs, in all uarm climates and southern United States. The dogs show respiratory

difficulties.

The filariids infecting wild animals are species

of Brugia, Loa, Dipetalonema, Onchocerca and Dirofilaria.

Tuo species of Erugia are parasitic in uild

animals. B. malayi, in tigers, slou loris and ant-eatera

in India, Halaya, Orissa and Kenya and B. pahangi in monkeys, slow loris, tigers, other uild felines and

ant-eaters in Plalaya.

Loa loa is found in monkeys in Belgian Congo.

One species of Dipetalonema, D. perstans is a

common parasite of apes in rain forests of Uest and

Central Africa and in chimpanzee in Belgian Congo. Species

of Onchocerca are found parasitic in antelopes and one

species of Dirofilaria, D. tennuis is found to infect

r accoons.

The speciation in parasitic nematodes is usually based on morphologi cal characters of adults but in filarial parasites, adult is hard to find and the only available

stage is microfilariae, often present in blood smears made at night. The diagnosis of most human cases of

filariasis is based on detection of microfilariae in peripheral blood. Conseguently attempts are made by uorkers to find relatively simple and reliable methods of differentiating the species of filarial uorms by identifying microfilariae present in peripheral blood. 6

riorphologically, the cuticular structures like cephalic papillae, general body surface and caudal papillae are of great ualue for distinguishing the different filarial infections. In microfilariae usually the nuclear characters and presence or absence of sheath is taken into consideration for identifying the genus and species of filarial uorms. Uhen these characters are not sufficient enough, the cephalic structures of microfilariae (hook and spines), the pharyngeal thread and Innenkorper provide good taxonomic tools.

The histological features like musculature, nuclear constancy of oesophagus and cells of intestine are of great value in . If the change in histological structures is studied after treatment uith certain drugs, it may give an important clue for treatment of filariasis, for example the drugs may form plugs over orifices, they may develop sterility in uorms, etc.

The histochemical tests nrovide a neu taxonomic tool for distinguishing the species of filarial worms by localizing the phosphatases, sulphatases and esterases in microfilariae. These are very -specific in their distribution in different species.

The iji vitro studies give a chance for studying the life cycle stages outside the body of vector and every detail can be studied without interference uith host tissue. I n vitro studies orov/ide 3 chance to test the effect of

neu drugs on parasite only. Later the toxicity tests

can be made in expprimental animals as the nsu drugs can

never be tested on human subjects directly.

The techniques for diagnosing human filariasis and

classifying them into different qrouos are not very

advanced. The morphological, histological and

histochemical tests for different species are not very

advanced, and the studies on ijn vitro cultivation are

also quite scattered ?nd inadequate. The crou filaria,

Chandlere11a haukinqi found in the right auricle of heart of Corvus macrorhynchos (Uagler), provides a very

good opportunity for all such studies. Hence this uiorm

has been chosen as a model snKcies for all above mentioned studies, as it is homologous with human filaria. 8

HISTORICAL REWIEU

1. (Morphology

(a) Light microscopy

(i) Adult

The genus Chandlerella was proposed by Yorke and Maplestone (1926) for Pilarja bosei Chandler, 1924, uhich uas described from racket tailed rjronqo

(Oissemurus paradiscus), Cory us crone , Pica pica and

Certhia familiarjs from the Zoological Gardens,

Calcutta. Later Pandit, f^enon and Iyer (1929) also described this species from right auricle of a emu in rnadras. Gilbert (193D) described a second species

C. stantchinskyi from India uhich uas also described by Sultana (1962) from the heart of a uhite backed munia (Uroloncha striata striata). Li (1933) described C. sinensis from lungs and trachea of

Corpus sinensis an'H Urocissa sinens is in China.

Tubangui and f^iasalungan (193?) described

C. lepidoqrammi from Philippines from L^pidogramnius cumingi. Tr lo ff (1) described £. lienalis from fxussia from horse. Ali (1957) recorded C. sinensis and added one more species C. singhi fnm the heart 9

of Corvus macrorhynchTS in Hyderabad. Yeh (1957) described a neu snecies C. braz iliens is from green billed touchsn frnrr . Rasheed (1960) reported

C. Columbiqallinae fAunustine, 19'37) from Crncnpus phaenicoptercis, and described ine neu species

C. thapari from 3cxicola torquata from Hyderabad.

3ones (1961) described f lexiuaqinaIj s from

Coruus brachyrhynchos from nontqomery country, Qhio.

Sultana (1962) added three more spc5cies to this qenus

C. himalayansis from Himalayan kestrel (Cerchneis tinnunculus inte rst inrtus), C. bucklevi from yellou fronted pied uoorioecker (Leiopirus mahrattensis blanforrii"! and C. a]ii fnm large pied wagtail

(Hotacilla maderaspatonsis) from Hyderabad.

Chatterjee, Son and Bhattacharya (1965) described the nresent species ChandlerelJa haukinqi from heart of

Corvus mocrorhynchos from Luckoou.

'ii) 'Microfilaria

Since the adult filarial uorms are difficult ti obtain the speciation or diagnosis of these uorms is possible only by characterizing and distinguishing microfilariae of different species present in osriphural blood. 10

Fullcborn (1Q13) 3tu'^iod the morpholoqical difPerencos in differunt tnicnf ilariae. Feng (1933) made a comparatiwe study of the microfilariae of

Brugia malayi and UuchBroria hancrofti and found the microfilariae of 8. malayi to be twisted in appearance.

Iyengar (1939) also studied the differences in morphology of microfilariae of U. bancrofti and n. malayi and found a crinkled appearance and uauy contours in B. malayi. Uilson (1956) gave a Feu more characters of microfilariae for distinquishinq these tuo species. The microfilariae of B. malayi are shorter than those of U. bancrofti and are usually twisted uith curv/es and kinks, whereas microfilariae of U. bancrofti are lonqer, smooth and with few curuos. The nuclei in 'nicrof i lari ac of

E. malayi are smudqed and overlapping but they are discrete in U. bancrofti. Therj are two nuclei in tail of malayi which arc absent in U. hancrofti.

Chandler and l\uari (1960), in their text book

'Introduction to Parasitology', identified UuchDreria hancrofti, Druqia malayi, Loa loa, tJ inetalonrma nerstans, Plansonella ozzardi. Onchocerca uoluulus and Dirofilaria i^nitis by distinguishing thriir microfilariae on the basis of orescncc or absence 11

of sheath and differr.ncos in nuclear land marks.

Roth^tein and Broun (1960) separated different

microfilariae on the basis of nuclear characters, using

v/ital stains. Taylor (1950) studied .the structure of

microfilariae of Loa loa, Uuchereria bancrofti,

Oirpfilaria immitis, D. rspens and D. aethiops.

SchPcher (1962) studied in detail the structure

of microfilaria of Brugia pahangi and described a feu

possible features for its rapid differentiation from

microfilariae of B. malayi and B. patei. Schacher,

Geddaui and Churchill (1967) studied the nuclear

number of microfilariae and proposed their number as

a test for intraspecific groupings and evolution in

Uuchereria bancrofti found in different geograohic

areas. Schacher and Geddaui (1969) again presented

an analysis of speciation and evolution in Uuchereria

bancrofti by the study of nuclear constancy (eutely)

in microfilariae.

Pnartinnz Baez (1176) described the microfilaria

of Inchocurca volvulus. He gave spccial att.jntion to

cephalic and caudal extremities and to some of the

cells in major part nf the body. He recommended similar studies in other endemic aroas so as to search the possible dif ferenc-.s , 12

Rpqprding cephalic and pharyngeal structures and Innonkorper, uery little unrk has been done only in recent years.

As fi^^rly as 1966, Sivanandam and Frednricks distinguished the microfilariae of Brugia pahangi and

B. malayi on the basis of Innenkorper. Laurence and

Simpson (1968) studied the cephalic hook and spines of microfilariae of Brngia, lu'uchereria, Loa,

C ardiofilarj a ^ Onchocerca, , Uipotalonoma and

Litomosoides and found these characters to be very specific for different oenera. Laurence and Simpson

(1969) again stained cephalic structures, oral ring, pharyngeal thread, Innenkorper and some other important structures like excretory pore, pores of SchuanzgebiIde in microfi lariae of L/uchereria, Brugia, Loa and

C ardiofi]arja. Again Laurencc and Simpson (I97l) described all the structures present in microfilaria of Brugia, the sheath, the cuticle, the cephalic space, the nuclear colur^^n, the sub-cuticular cells, the nervf ring, the excretory pore, the nharyngeal thread, the

Innenkorper, the cell, the cells, the anal vesicle, the SchuanzgobiIde and the tail nuclei.

Reaarding tuo forms of microfilariae, KorkL

( 1 929) described the frorpholoqy of typiral and atypical 13

forms of microfilariae in Uuchererja bancrpfti. He

(192n) addud that atypical forms were much shortpr and slightly thickor. The length of cephalic space was shorter, the nuclei overlap, the nerv/e ring sliqhtly anteriorly placed and tail is abruptly drawn out. Schacher (1962) working on B. pahangi and

B. malayi showed that the body length of microfilariae is highly variable and hence cannot be taken as criteria for species diagnosis. 14

(b) Stereoscan study

(i) Adult

Although microtopographical features of the head region and the surface structure of body cuticle hav/e a great significance in taxonomy, yet very little uork has been done to study the finer details of these structures by stereoscan studies.

Allison, Ubelaker, Uebster and Riddle (1972) described a simplified technique for preparation of helminths for scanning electron microscopy. Tf^isystated that the specimens have to be treated in a manner for making them self supporting in high vacuum and for their surfaces to carry off any charge that biiilds up due to electron bombardment. In this uorkthey studied the anterior region of Sulcascaris sulcatum.

Dick (1972) studied the ultra structure of cuticle of Syphacia obyelata. Ueise (1973) studied the surface topography of cuticle, anterior and caudal ends and the male and female reproductive system of

^'Temonchus contortus by scanning electron microscopy.

Dick ^nd Uright (1974a) studied the ultrastructuro of the cuticle associated uith male reproductive structures in nematode Syphacia obvelata. They found variations in the cuticle of ventral copulatory structures, cloacd papillae, clo'ica, spicules, spicular sheath, gubern^culum and other accessory structures. 15

Dick and Wright (ig74b) i n a separstB papor

studied the cuticular variations associated uith excretory pore, vulva and vagina vera. Arizono,

natsuo and Yoshida (1976) did scanning olectron

microscopy of Strongyloides planiceps. They studied the external features of parasitic and free living

adult females, free living males, infective and first stage rhabditoid larvae. Hogger, Estey, Kisiel and

Zuckerman (1976) studied the differences in cuticular markings betueen young and old females of

Caenorhabditis brigqsae. Kikuchi (1976) in his three separate papers studied cuticular morphology of

Ascaris lumbricoides and compared it uith A. suum,

the cuticular morphology of Ascaridia columbae and r-ffi • the cuticular structures of Terranova decipiens and

Gnothostoma doloresi. Lopez C aba Hero and Cast an ys

Cuello (1 976) studied the finer details of cephalic extremity of Splendidofil^ria mavis (Leiper)

C^nnert, 1937 uith storeoscan electron microscope.

Setasuban (1976) studied the cuticular details of

Bathmostomum sangori Cobbold, 1879 under stereoscon electron microscope. Uang and Fujita (1976) compared the external morphology of Ascaris suum and

A. lumbricoides uith scanning electron microscopy and found no difference in the external morphology of these tuo. 16

Barus, Kotrla and Tenora (1977) did scanning electron microscopy of spicular sheath of some trichurids. Shava and Leuis (1977) studied the cuticle of oxyurid nematodes belonging to genus Syphacia.

Shoho and Uni (1977) studied the external features of Setaria digitata^ S, marshalli, S.m. pandei,

equina and 3. labiato-papillosa under stereoscan electron microscope and amplified the description of these species in the light of stereoscan studies.

They also discussed taxonomic importance of these characters. Uertheim and Chabaud (1977) studied the cephalic characters of the entire family

Pneumospiruridae (Thelozioidea - Nematoda) and in the light of these characters, they revised the entire family.

r^archiondo and Sauyer (1978) made a stereoscan study of microtopoqraphical features of head region of

Physaloptera felidis. Tenora, Uiger and Barus (1Q78) did scanning electron microscopic studies on three species of the genus Syphacia, S. obvelata, 3. niqoriana and 3. stroma. They pointed out similar transverse striations on the body but difference in the structure of htjad. Uigcr, Barus and Tenora (1 978) studied the ultrastructure of head and surface structure of the cuticle of Syphacia petruseuizi, S. nigeriana,

3. frederici and S. stroma. They distinguished these 17

species from each other on head and cuticular structures. Uong and Brummer (1978) studied the cuticular morphology of five species of Dirofilaria,

D. immitis, 0. corynodes, D. magnilarvatum, D. repens and D. tennuis. They also observed variations in patterns on en-face view of head and variations in the cuticular patterns uith the portion and aspect of the uorm examined. Barus, Uiger, Tenora and

Stanek (1979) observed the lip denticles of Toxascaris - •' ' mi leonine, and T. cati under stereoscan electron microscope,

(ii) Microfilaria

Regarding the ultrastructures of microfilariae, very little work has bee;n done. McLaren (1 972) studied the ultrastructure of the microfilariae of Dipctalonema vitoaL, D. setariosum, Oirofilaria immitis, Loa loa and

Litomosoides carinii. U'Leary (1972) studied the ultrastructural aspects of microfilariae of Oirofilaria immitis as reconstructed from serial sections.

Tongu (1974) studied the ultrastructure of microfilariae of . nieqevillc, Marjolet and Ucrmeil (1978) observed the microfilariae of

Dipetalonema viteac under SEM. Thoy found 2 types of 18

microfilorlae in blood of hamsters. These differ in form of anterior end. The less numerous type has a long, fairly thin (about 2 yum) neck made up of 12-14 regular cuticular annules. In other typo, neck is shorter and made up of 6-8 annules. 19

2. Histology

Not much work on histology of nematodes has been done. Whatever has been done is in Form of scattered papers and confined to only a feu species. Schacher

(1962) studied the histology of different organs of

Brugia pahangi, a filariid of cat, at different important levels. Later Ansari and Basir (1964) made a comprehensiue study of histological characteristics of every organ and system of Set aria ceryj, a filarial parasite of bui^'falo. Rest of the uorkers studied the histology of isolated parts in some or the other uorm.

V/on Siebold (1838) studied the complex nature of the cuticle in . A similar pattern was observed by de nann (1886) in the cuticle of Enoplus communis. Goldschmidt (1906) described

9 layers in the cuticle of Ascaris. Martini (1912) studied the cuticle of Oxyuris and found 9 layers in it. Schacher (1962) did not go into the detailed structure of the cuticle of Brugia pahangi and he simply described tuo layers in it, a cortical delicate basophilic layer and a fibrillar rafractile eosinophilic layer. Ansari and Basir (1964) described 8 cuticular layers in Setaria cervi. Inglis (1964) studied the structure of nematode cuticle. Asadov (1973) worked on the cuticle of Trichuris ovis. He observed 6 layers 20

in the cuticle. Magqnti (1979) studied the cuticular

strata in Enopli

based on cuticular strata in nematodes as compared to

Enoplia model.

Uieus differ regarding the origin of the cuticlo e

also. nijller (1929) described it as a secretion

product. Chitu/ood (1936) belieued that various layers

of the cuticle develop as protoplasmic condensation

from the living cell. Ansari and Basir (1964) agree

uith Chituood's vieu.

The cuticle is followed by sub-cuticle which is the middle layer of the body uall. itouart (1906)

called it epidermis, uhile studying anatomy of

Oncholaimus. There are different views regarding its origin. Hamann (1895) regards it ectodermal whereas zur jtrassen (1904) believes it to bo mesodermal in origin. Schacher (1962) did not go into the details of the origin of this layer and he simply described its thickenings at dorsal, ventral and lateral chords.

Ansari and Basir (1964) disagree with the terminalogy

and origin of this layer and called it sub-cuticula.

The importance of musculature in nematodes was first of all emphasised by Schncider (1860). He suggested that thu form, number and arrangement of muscle cells are of fundamental imoortance in the classification of nematodes. On the basis of these 21

characters ho proposed the tnrm platymyarian and

coolomyarian. The nematodes having fibrillar portion

of cells flat towards the body cavity are platymyarian

and those having fibrillar portion of cells bearing

groove, distally making the sarcoplasmic part to dip doun into and between the contractile layers, present on either side of the cells are coelomyarian. Further, he took into consideration the number of muscle cells in each sector, if feu, he called it meromyarian, and if numerous, polymyarian. He stated that meromyarian forms are platymyarian, and polymyarian forms are coelomyarian. Schneider's uork lost importance when

(Martini (1916) found that polymyarian and coelomyarian nematodes are meromyarian and platymyarian in first larval stage. This proves that polymyary and coelomyary is reached in later stage of development.

Schachcr (1962) described musculature of Bruqi a pahangi to be coelomyarian, not separated by dorsal and ventral chords. f^uscle cells in gravid females are compressed to thin dense layer uith nuclei among fibrillar elements.

Ansari and Bnsir (1964) described musculature of Setaria caryi to be polymyarian and coelomyarian type, broken at four pl^ccs by dorsal, ventral and tuo lateral chords. They described specialised muscles 22

supporting oesophagus, intestine, uulva, anus and

SDicules, Hirumi, Raski and Gones (1971) studied the morphological aspects of platymyarian and shallou coelomyarian muscles in Trichodorus christjei and

Longidorus elongatus (plant nematodes). Bogoyav/lens kii and Skrjabin (1971) made comparative histological investigations of the somatic musculature of certain nematodes of the sub-order spirurata.

Regarding the body fluid, Stewart (1906) s described a jelly like sut^tance having nuclei in it.

Schacher (1962) did not mention anything about body fluid. Ansari and Basir (195A) described a granular body fluid in Setaria cervi. They suggested that this fluid is necessary as is absent and this fluid may serve as haemocoelomic fluid of other invertebrates.

The connective tissue in nematodes has been described by Schneider (1902) under the term

" Bindegeuebe" . Looss (1 905) called it "strand like organs'" and Goldschmidt (1906) proposed the term

" I solationgeuebe" .

Schacher (1962) uhile studying histology of

Brugia pahangi did not mention anything about connective tissue. Ansari and Basir (1964) described nucleated strands between oesophagus and muscle layer in

5. cervi. 23

Uork on histology of oesophagus of nematodes is

scattered. f^artini (190S) and Chituood (1 930) studied

the oesophagus of nematodes and described that the

oesophagus is covered externally as well as internally

by a membrane called 'tunica propria' and regard it

to be the secretion of marginal nuclei,

Schacher (1962) described tuo oarts of oesophagus,

the muscular and the glandular in B. pahangi; the

muscular part is deeply eosinophilic uith large nuclei

and triangular lumen and the glandular part is granular,

vacuolated uith many gland and supporting cell nuclei.

He described the presence of a bilobed oesophago-

intestinal valve, histologically similar to oesophagus.

Ansari and Basir (1964) also described tuo

parts in oesophagus of 5. cervi, externally covered

by 'tunica pro.pria', uith triradiate lumen, supported

by special muscles callnd the somato-oesophageal

muscles. They also described oesophago-intestinal

valve, through uhich oesophagus opens into the intestine.

Looss (1905) considered the oesophago-intestinal value as a product of differentiation of intestine. Plagath

(1919) described it as a specialised part of oesophagus.

Chituood (1931) proposed the term oesophago-

intestinal v/alv/e. Onmori, Yoshimura and Ishigooka

(1976) did a comparative study of oesophageal lumen 24

in stronqyloidea. They presented the cross sections of oesophageal region of 13 species of Ancylostomatidae,

6 of 3trongylidae, one of Stephanuridae and §'of

Trichonematidae, and rccognised 5 types of oesophageal cuticular linings. They suggested that structure of oesophagus in cross section allows the differentiation of species. Sood and Sehajpal (1979) studied the oesophagus of H. contortus and found the lumen to be lined uith cuticle.

The histology of intestine of nematodes has also not received much attention. Chituood (1930) gave an interesting method for classifying nematodes depending on the number, the form, the height and the shape of cells in cross section and grouped the nematodes into different categories. Those having large number of calls are termed as myriocytous and those having irregular lumen are anisocytous.

Schacher (1962) described intestine to be made up of a thin hyaline outer membrane and a basophilic granular epithelium containing 5-9 nuclei per section at mid region. Ansari and Basir (1964) while describing histology of S. cervi gave their opinion that this effort of classification is uneven and troublesome. They described the intestine of cervi to be myriocytous and anisocytous. They suggested that myriocytous nematodes are always anisocytous. 25

Bogolepova (1975) studied the histological

characteristics of intestinal structure of Arnidostomum

anseri, Uncinaria stenocephala and Oesophagostomum uenulosum. Bogoya\ylenskii (1976) studied the

histology of fine structures of nematodes. Sood and

Sehajpal (1979) found intestinal epithelium prouided with developed brush borders in H_. contortus.

Uieujs among workers differ regarding uall of the intestine which consists of a single layer of epithelial cells, prov/ided uith conspicuous lining on the internal surface knoun as 'Stabchensaum' or bacillary layer.

DaegerskideId (1894) found that bacillary layer known as 'Stabchensaum' internally and the outer epithelial cell layer of the intestine consists of long separate rods. Looss (1905) described

'Stabchensaum' as cuticular layer. Quack (1913) made a detailed study of the intestinal cells and concluded that the rods described by 3aegerskioeId

(1B94) are longitudinal series of alv/eoli and inter- alveolar substance. Hetherington (1923) stated that sub-bacillary layer consists of fine granules which appear to be connected with each other and with the rods which form the bacillary layer. I^ueller (1929) suggested that bacillary and sub-bacillary layers 26

constitute neuromotor system in Ascaris. Chituood

(1930) explained the nature of bacillary layer and gave three possibilities.

1. Bacillary layer is a dewelopmont of minute

tubules uhich aid in absorption or excretion.

2. It is a secretion product of protective

nature.

3. It is a layer of amalgamated degenerate

cilia.

Ansari and Basir (1964) described bacillary layer consisting of cilia like rods. The rods are usually fused together forming a membrane, but at places they are separate. They found this layer to be high in cardiac part of intestine but lou in rectal part. There, is also a granular sub-baci llary layer or

'deckschicht' cor responding to the layer of basal granules in ciliated epithelium uhich is followed by external protoplasmic zone of intestine. They also described the basal lamella piesent outer to the protoplasmic zone.

Only Ansari and Basir (1964) described an intestino-rectal valve surrounded by sphincter muscles.

They described the rectum to be lined by cuticle and supported by special muscles, depressor ani and dilator ani. 27

Uorkors like Eberth (1363), Hamann (1395),

Ehlers (1899) and V/oltzenloge 1 (1902) described rectal glands. Looss (1905), while studying

Ancylostoma concluded that these are made of connectik/e tissue. Martini (1913) also believed that these are the rectal ligaments. flagath (1919) took them as sarcoplasm of the sphincter muscles, as none of these workers could see the duct from these structures leading into rectum. Chituood (1930) uas first to observ/e the orifices of rectal glands in Rhabditis, f^acracis, Cephalobe llus and Hystringnathus. Baker

(1936) confirmed Chituood's observations in Heterakis.

Schacher (1962) did not mention anything about these structures. Ansari and Basir (1964) though observed these structures in Setaria cervi but could not conclude their nature as they were unable to observe any duct or any orifice from these structures. Only

Ansari and Basir (1964) studied the histology of the cloaca jf male in S. cervi.

The histological anatomy of female reproductive system was first studied by Chitwood (1929) where he described ovaries to begin with a cap cell, which covers the blind end of ovary and a part of ovarial epithelium. Musso (1930) gave a completely different view and regarded the cap cell to be undifferentiated germinal stem coll which gave rise both to epithelial 37

and germinal culls. Schacher (1962) described tuo

Tuaries, opisthodeIphic with germinal and growth zones and a terminal cap cell. In 3etaria cerui

Ansari and Basir (1964) stated that ovary is divisible into tuo zones, the germinal zone followed by growth zone. The tip of the germinal zone is covered by the apical cell.

The histological anatomy of oviducts was fjrst studied by Schacher (1962) in Brugia pahangi. He observed the oviducts with narrow lumen surrounded by thick cuboidal epithelium of about 6 cells per section. Later Ansari and Basir (1964) described the histology of oviducts of Setaria cervi, having narrow lumen surrounded by cuboidal cells. Onushko

(1974) studied the histological structure of female genital organs of Syngamus trachca at different stages of post-embryonic development.

Different workers put forth different views regarding ^hu origin of rcceptaculum seminis. "'lagath

(1919) believed it to be part of oviduct. Chitwood and Chitwood (1933) described it as a part of uterus.

Schacher (1962) stat(>d that the walls of seminal receptacle are thick made of high cuboidal cells. He did not mention its origin. Ansari and Basir (1964) believe that seminal receptacle is a modified part of uterus. 29

Schacher (1962) dDscribod the histology of uterus in B. oahangi where th ^ uall is tuo layered consisting of an outer musclo coat and an inner epithelium of tall cells, Ansari and Basir (1964) also described the histology of uterus of Setaria cerui where the ualls consist of outer muscular coat of circular muscles and inner layer of large, columnar, nucleate cells.

Schacher (1962) described the histology of vagina in B. pahangi. He divided it into tuo regions, vagina uterina and vagina vc/ra. Vagina uterina has a thick outer muscle coat and an equally thick inner layer of cuboidal colls whereas vagina \je^Ta is made of several layers of circular and oblique muscles.

Ansari and Basir (1964) also described the vagina of

Setaria cervi having the same characteristics. The details of vulva have been described for the first time by Ansari and Basir (19G4) in Setaria ccrvi.

5ood and Knur (1977) studied the histology of vulva of

Haemonchus contortus. They found an outer colourless and innur thick sclerotisud layer with small protoplasmic core. The extra swellings are formed by cuticle.

Mo other worker jjrlier than Schacher (1962) studied the histological details of male reproductive 30

system. HG statod TNO tubular solid testis uith two zones, the qGrminal and th^ growth zone in Brugia pahangi. He observ/f-d thy testis starting as a single ccll, folloucd by a suminal vusicle uith walls of variable thickness. It is basophilic and leads into the vas deferens uith ten nuclei per section. The vas deferens is followed by a muscular ejaculatory duct which opens outside the body through cloaca.

Ansari and Basir (1964) described similar characteristics of male reproductive system in Set aria ceryi.

Schacher (1962) dcscribcd the capitulum of left spicule to bo tubular and right spicule beginning with thick hollow tube with a medially directed ala, the lumen is gradually shifted and reduced to form small channel in B. pahangi. Ansari and Basir (1964) described two unequal and dissimilar spicules in

Setaria cervi. The left spicule is larger than right, having long pitted shaft followed by tapering blade.

Tht. blade is flanged assuming gutter shape. The right spicule is stout, curved and boat shaped. They described thu soicules to be covered by a cuticular layer h.vinq a protoplasmic core. ai

3. Histochemistry

(a) Acid phosphatase

As early as 19^6, Chourihury, Dasqunta, Ray and

Bhaduri studied the histochetiica 1 pattern of microfilaria of Liuchereria bancrofti and found acid phosphatase to be absent in the Innenkbrper,

Pitithory (1T66) uas first to report the enzyme activity differences in micr^fi]ariae jf different species. This provides a possibility of a neu taxonomic tool for identifying the filarial uorms.

The feasibility of separating filarial worms has subsequently been deminstrated by Chalifoux and Hunt

(1971). These unrkers shiued strikingly different patterns for acid phosphatase antiuity in microfilariae of

Oirofilaria immitis and Dipetalonema reconditum even in mixed infections.

Balbo and Abate ^1972) not only cnnfirmed the observations of Challfojx and Hunt hut reported a third distinct pattern for Oirifilarir^ repens. Chalifoux,

Hunt, Garcia, Sehgal and Comiskey (1^73) went still further for classifying the different filarial uorms.

They combined acid phosphatase activity uith the information on lemth and prencs of sheath to compose a dichotomous key identifyinn 11 snecies of microfilariae from the neu world mnkeys. 32

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

differentiated the microfilariae if Bruqia pahangi and

sub-periodic B. ma layi by localizinq the acid phosphatase

activ/ity.

Teruedou and Huff (1976) studied the acid

phosphatase activity in microfilariae of Uuchereria

bancrofti . Braun-flunz i nqe r and Southgate (1977) studied the acid phosphatase activity in microfilariae of

Onchocerca uolvulus, causing onchocercJasis in Uest Africa,

Four distinct patterns of enzyme staining uere observed.

They concluded that a number of biological strains or variants of Hnchocprca volvulus co-exist in Uest Africa and suggested for further research that could result in the practical application in onchocerciasis control programme. G'^ar (1977) studied the distribution of acid phosphatase in larval stages of IJuchereria bancrofti,

Brugia malayi, B. pahangi and Dirofilaria immitis in mosquito, and sunqested that an accurate diagnosis of these genera could be made on the basis of pattern of distribution. ']mar and Kuhlou (1977) diffnrentiated the microfilariae of Loa loa and iJinetalonema pcrstans from Cnmeroun on the bfsis of ncid phosphatase activity.

Omar (1978) performed hi s tTChei ca 1 differentiation in microfilariae of Inchocerca volvulus by localizing acid phosphatase, in IJn 5t African rain-forests (Liberia) 33

Sudan-savana (Upper Volta), Guatemala and Yemen. He distinguished 5 distinct staining patterns, Frequently present in a single infected person. Aaeinn, strain difference or other factors uere considered to be causing these differences, 'Irnar and Schulz-Key (1978) studied the acid phosphatase activity in larval stages of Onchocerca volvyulus developing in the v/ector,

S imu lium dam no s urn. l/oen and Blotkamp (197 8) studied acid phosphatase activity in microfilariae jf Oipntalonema,

Dirofilaria, Dnchocerca and Setaria spp. Th.3y added that staining pattern for acid phosnhatase activity may hn Ip in differentiation of microfilariae of different species within the same host. Yen and f'iak (197R) localized the acid phosohatase activity in microfilariae of

Brugia, Uuchereria, 0irofilaria and Breinlia. They concluded that acid ohosphatc^so activity in microfilariae gives sufficiently characteri3tic and consistent results for di f f e re nt i a'".i 3n of oven closely related snocius like Brugia malpyi and B. pahanni, Oirifilaria rcpens and D, immitis and Breinlia booliati from B, surqenti.

The enzyme rUstribution of Plalaygian and rural strain of b'. bancrofti uos different fr m those of othtT re gions. 34

Not much attontion has beun paid to the enzyme localization sf adult unrms. (^aki, Ito and Yanagisaua

(1977) studied acid nhospbatase m adult Dirofilaria immitis. f^aki and Yanaqisaua (lQgO) jbserund high acid phosphatase activity in Dinfilaria immit is at pH 3.Q-5.R.

The body uali and reproductive organs showed high acid phosphatase activity.

Among other nematodes Er]angur and Gorshon (1970) studied acid phosphatase activity in Turbatrix aceti.

Ruitonbrrg and LoenHorsloot (1Q71) Found acid phosphatase in intestinal tract and brush borders of larvae of

Anisakis sp. f^eznik ^971) localized acid phosphatase activity in the cutirln of Ascaridia qalli and Ascaris lumbricoides. Bolla, L'einstein and Lou (1974) worked on the acid phosphatase activity in developing and pqeing Nippostrongylus brasiliensis. They found nreatest activity in third staqo larvae and T-day post-infection adults. Seniuta (1975) localized acid phosphatase in deve lopmentt^l stages of Trichinclla spiralis. He observed that activity varied uith embryonal, migrating, developing muscular and invasivn larvae and intestinal uorms. Sharma, Singh and Sharna (1976) studied acid phosphatase in Stephanurus duntatus. They found maximum activity at pH 4.0, in orsoohc.gu3, intestine, ovary, 35

uterine uall, cqqs, cuticle and in the testis. f'laki and

Yanagisaup (1930) obsRrv/ed hioh acid phosphatase actiuity at pH 4.5-6.0 in Angiostrongylus cantonensis. Host of the activity uas localized in the body uall.

(b) Alkaline phosphatase

Choudhury, Dasgupta, Ray and Bhaduri (1956) localised a Ikaline phosphatase in microfilariae of

Uuchere ria bancrofti. They found Tnnenkorper to be highly positive. A feeble reaction l'3s observed in the cuticle and nuclei. R oy ch ludhury, Lahiri, Fenedle and Nabu ^ 975) reported alkalinp phosphatase cictivity in the vector

Aedes aegypti after feeding it on a dog positive for

Dirofilaria repens. Those authors also studied enzyme activity in the adult uorms and found it to bo greater in male than in female.

Ueen and Blotknmp fig73) studied the alkaline phosphatase activity in micnfilnriae of Oipeta lonoma,

Dirofilaria, nnchocerca find Setaria spp., and suggested that the patterns of enzyme activity may aid to the differentiation of microfilariae of different species uithin the same host.

Among other nematodes, Segidn (I97l) localized alkaline phosphatase in nerve cells of Ascaridia galli.

Seniuta (1975) studied alkaline phosphatase in developmental staqes of Trichinolla spiralis and found it to be absent. 36

(c) Adenosine trip'iosphafca'je

Only Couinriiiar, Cauande and Harinath (1 974) detected adinosine triphosphatase activity in homogenates of nicrofi lariae Liuchererid bancrofti. No other record in filnrial uorms is available.

Among other nematodes Pouyakel (116S) studied adenosine triphosph?tase activity in muscle tissue mitochondria of pig Ascaris. Ruitenberg and Loendersloot

(l971) found adenosine triphosphatase in cuticle of

Anisakis larvae. Reznik (1971) found AtPase in cuticle of both the species viz., Ascaridia galli and Ascaris lumbricoides. Bok (1973) studied distribution of adenosine triphosphatase in sorne hplminths. Hayashi (1973) detected adenosine triphosphatase formatiin by mitochondria from Ascaris lumbricoides suis. Pavlov

(1973) studied the role of adenn3ine triphosphatase in transport of amino-acids in -tscaris suwv. Van den Bossche

(1974) detected a low ,-mlecular cjntent of membrane bound adenosine triphosphatase in mitochondria of

Ascaris suum muscles. Heznik (1975) found magnesium- dependent adenosine triphosohatase in intestinal epithelium, medial nerve cord, somatic musculature, oesophageal and reproductive system of Ascaridia galli.

Seniuta (1975) localized adenosine triphosphatase in 37

developmental staqes of Trichine11a spiralis. He obsaryed that the activ/ity varied with embryonal, migrating, developing muscular and inva^iue larvae and intestinal worms. Ahn found adenosine trinhosphatasn activity in sub-cuticular syncytial cells, serial zone of intestinal epithelium and reticular cells of Ascaris suum. He also studied the uptake of '^^C ATP after 1 4 incubating the uorms in Tyrode solution containing C ATP and found that radio-active label uas highly concentrated in intestinal epithelial cells and sub-cuticular tissues.

Ualker (1977) studied the relationship between temp^^rature change and mitochondrial ATPase activity. (d) Carboxyl esterases

Hart and Lee (1966) localized the distribution of cholinesterase activity and inhibition by anthelmintics in various nematode pnrasiL<33. Ueznik (1 969) studied the distribution of o'-in-speci fi c oster-^ses in the body uall of Ascaridia galli. Sanderson (1969) studied cholinestrrasB activity in animal o^'r^sitic nematodes.

Erhnger d»d Crershon (1 970) studied cho li nestn rase activity in relation to the age of Turbntrix aceti. Reznik and

Didenko (1 970) localized the Ccrbmic estnrd3i?s in cuticular, sub-cuticu 1 ^ r nnd muscle, layers of the body uall and alimentary trart of ^-dult Trichuris niuris. 38

They found that non-cellular cuticle does not contain

carbonic esterases but the sub-cuticular layer and muscle

layer contain non-specific esterases and cholinesterase.

Non-specific esterases uere found in cells of alimentary

t r act.

Reznik (1971) found considerable amount of

carboxylic esterase in cuticle, hypodermis, muscle and

intestinal epithelium of Ascaridia qalli and Ascaris

lumbricoides. He fl97l) also worked on carbonic

esterases of 3 parasitic nematodes and found that

cuticle of Ascaris suum and Ascaridia galli contained

non-specific estprases 3nd that of Trichuris contained

cholinesterase. Cholinestnrase occurred in intercellular

spaces of gut epithelium of Ascaris suum and Ascaridia

galli but not in Goithelium of Trichuris. Ruitenberg

and Loonderslont (1971) examinr-d larvae of Anisakis sp.

and found non-specific est .rase activity in cxcrotory organ and suggested that this histochemical test may

be used for testing viability of larvae and offeet of

anthelmintics. They (1971) again studied the histochemical

nature of excretory organ of Anisakis larvae and found strong activity for non-specific esterases in latrral part of the organ and moderatr activity in central area.

Sanderson (I97l) localiBed the distributiin of cholinesterase activity in some species of Strongyloidea and Oxyuroidea. 39

Tan and Zam (iTTl) found non-specific esterases in pcrientric fluid of Ascarjs suum. Gupta and Sood

(1974) domonstratod non-specific esterase activ/ity in the body wall of Hetorakis nusilla --^nd discussed its functional significance. rialyutina (1974) studied the acotycholinesterase system in nerve and muscle tissues of Ascaridia palli. Sanderson (1974) studied acetyl- cholinesterase in IMippostronqylus brasiliensis.

Goh and Davey (1176) studied nervous system of

Phocanema Hecipens using acetylcholine method. They found acetylcholinesterase to be present ns small discrete granules in nerve ring and 6 longitudinal nerve cords. Ijright and Aunn (1 976) detected the cholinesterase activity in th^' nr-rvc ring in i^anrgrellus redivivus,

Cnenorhabditis cleqans, Prionchulus punctatus,

Aphelcnchus dvenau and TJitylonrhus dipsaci. They found that ultrasonic oretreatment of intact or cut nematodes improved qro;^tly the st<'ining consistency at the nerve ring. Yoat-3 and Jgilvio (1976) studied the enzymic properties nnd possible physiological role of acetyl- cholinesterase in Nippostrongylus brasiliensis and

Necator americanus.

Nuosu (197S) studied age related changes in esterase and acoty1cholinesternse activities of the infective larvae of Ancylostoma tubaeforma. Dang, Ning 40

and Uanq (1979) detbrmincd cho linestL^rase activity in

aui3r 2000 intect Ancylostoma duodenalo and over 1000

Necntor amoricjnus rccovGred Fnm 2 05 patients and

treated uith one nf thfj SGV' ral ant ho Ini nti cs . Terenina

(1979) studied the cholinnstirase activity in female

genital tracts of 7 species of nematodes in different

sub-orders. The enzy^ie uas active in areas of muscles

and ova.

(e) Aryl sulphatase

This enzyme has mt so far been recorded in nematodes. Anonq othf r he I^m i nt iis Bogitsch (1 966) studied aryIsulphotase in Int^jrmediate layer of cysticercoid. 41

4. ^ \/itro cultiv/ation

Attempts by workers to cultivate ^ ^/itro, the

microfilariae of different filarial uorms, have met

LJith little success. Initially the microfilariae were

cultivated in uhole blood. In fact the cultivation

in vitro of microfilariae of the filarial nematodes

parasitic in uarm blooded hosts uas first successfully

carried out by Hobson (1948). This uork ujas reviewed

by Earl (1959), uho carried out a series of iri vitro

experiments on the maintenance of the microfilariae of

Dirofilaria immitis, the dog heart worm, in a variety

of media at 37 C. The microfilariae survived for

4 days in medium 199 alone but uihen supplemented uith

10% inactivated dog serum, the microfilariae survived

for 43 days at 37 C; they survived for 61 days when

serum supplement uas increased to 30%. He obtained

similar results uhen supplemented medium 199 with ox or

horse sera. The results did not improve by using

kidney tissue culture overlaid uith medium 199 + 10%

inactivated dog serum (survival time 40 days only).

Taylor (1 960a) cultivated ijn vitro, the microfilariae of D. immitis in a variety of media and

cultures of mosquito uhole gut. She used medium 199 supplemented uith horse serum, raw liver or mosquito extracts, rod blood cells -jr dilute Tyrode's solution at 22 C, in an attempt to induce development of the 42

mosquito stages. The micnfilariae survived for 14 days, no grouth or development occurred. She (1950a) maintained ^ vitro, the microfilariae of Uuchereria bancrofti and Loa loa for 10-14 days, in a variety of media containing serum, blond cells and some chemically defined components. The microfilariae of U. bancrofti exsheathed but no development occurred uhereas there uaa an indication of the first stage of development in microfilariae of Loa loa. In that she observed, the

G cells 1 and 2, to have divided.

In the following year 1961 Sawyer and Ueinstein attempted to determine the importance of various inorganic ions for vitro survival of D^. immitjs microfilariae. He tested the inorganic components of

Earle's, Gey's, Krebs-Ringer-phosphate and Locke's solution. In modified Locke's solution, the microfilariae survived for 35 h at 37 C and 55 h at

27 C. In Krebs-Ringer-phosphate uith 0.25^^ glucose and 1.5;?^ sodium caseinate, buffered at nH 7.2, the micnfilariae survived for 5 days at 27 C but only for 40 h at 37 C.

This uork was later extended by Sauyer and

Ueinstein (1 961, 1 963a,b,c). They obtained the development of microfilariae to the late first stage

(early 'sausage stage') in culturt:s of heparinated blood at 22 or 27 C. This development occurred as 43

early as 3 days if culture but no further development occurred throughout the 21 day culture period. They cultivated in vitro, the microfilariae of Oipetalonema in the some manner and obtainu^i 'sausage stage' aft^r

12 days.

Sauycr and Ueinstein (ig63b) incubated the microfilariae of D. immitis at 22 or 27 C in human blood or in rabbit blood or in Locke's solution containing a suspension of dog blood cells. Tho development to 'sausage stage' uas obtained in these media. They also observed the survival of microfilariae for several weeks Liithnut development in dog plasma or horse serum. In chemically defined culture medium

(NCTC 109) they survived only for 7 days and no development occurred. Sawyer and Ueinstein (1953c) went on to find the optimum concentration of horse serum which would promote the growth to the 'sausage' form in NCTC 109. They obtained the maximum yield of

30^ at 27 C with 5-10f inactivated serum in NCTC 1G9 and the survival tine increased to B days. A definite increase in width also occurred in less than 30 h of incubation. They found that gassing the cultures every 2-3 days with 5/'. CO2 in air was beneficial but the medium was not changed during culture. The microfilariae did not develop at 37 C and the survival time was reduced ti 4 days. 44

Weinstein (1963) applied his technique for

cultivating microfilariae of Litomo3oides carinii.

Exshe^thnent uas induced by lytic enzymes omitting

saponin which uas tixic. 'Sausage stage' uas obtained

in 6 days in NCTC 109 uith 20% inactivated human serum

and antibiotics, supolemented uith amino-acids, suaars

and organic acids of Grace's medium. Here also he

found that the gassing air uith CQ2 uas beneficial.

Sauyer and Cheever (1962) reported that

microfilariae found in Columbian marmoset (Oedipomidas)

survived for 22 days in a suspension of marmoset

kidney cells in medium 199 + calf serum. During this time microfilariae shoued an increase of 25% in length but this development uas supposed to be abnormal as

normal development to sausage stage is accompanied

by shortening and fattening process.

Ueinstein (1 963) cultivated ijn vitro the microfilariae of Uuchereria bancrpfti in the same manner

as microfilariae of Dirofilaria immitis and obtained

'sausaqo stage' in 4-7% of microfilariae. He added

additional amino-acids, sugars and organic acids to supplement the medium, 40% of the microfilariae shoued signs of development Jj]_ vitro. The time of Jevelopment uas considerably prolonged as compared to the time required in mrmal hist. 45

Plost successful cultivation of microfilariae has been achiev/ed by Uood and Suitor (1966) using microfilariae of i'^acacanema formosana from the blood of Taiwan monkey Hacaca cyclopjs. They used Aedes aegypti cell lines overlaid uith a medium containing

0.5^ haemolymph from Philosamia cynthia + 10^ foetal bovine serum in Grace's insect cell culture medium GHA,

Plicrofilariae uere separated from heparinated blood by treatment with saponin before being cultured. The development to third stage took place at 22 C but not at 28 C. [Medium uas not changed throughout the 33 day culture period. Liithin 5 days the larvae had reached

'sausage stage' and the movement uas maximum. Between

6-9 days larvae grew raoidly in length and narrowed slightly and in 14-19 days the larvae reached the infective stage.

'Jcinstoin (1970) reported the develipmont of

Dinetalonem" • a reconditu, m microfilariae to 'sausage' f )rm using uholc blood and blood fractions from ;iog and other mammals.

Aiki (1971) reported active exsheathment of

Uuchereria bancrofti and Brugia pahangi microfilariae in thick blood films, kopt in moist chambers and on agar plates at different temperatures, salinities and oH. At 15-20 C, more than 50% microfilariae of 46

U. bancrofti exshGathed in 4 h, 80% in h and 90% in

12 h. On agar platGs 90% nf U. bancrofti microfilariae

cxsheathed uithin 8 h and 98.2^ of B. pahangi did so in

1 h. The rate declines at higher temperature. Ib'hen

agar plate cultures uere exposed at 20 C follouinq 8 h storage at 5 C the exsheathment rate uiaa accelerated.

Lack of saline or concentrations ouer 2% killed the microfilariae. The best concentration was D.9% and optimum pH uas 5.8 - 6.8.

Cupp (1972) uorking in a Uorkshop on Dev/elopment of Filariac in Hosquitoos, U.S.-Japan Co-oporativ/e

Medical Science Program, University of California,

Los Angeles, has reported development to late L^ stage in D, immitis and D. corynodes in mosquito cell

lines uhich appeared enhanced by insect haemolymph.

luamoto (1972) made extensive jji vitro

Gxperiments with Dirofilaria immitis and Uuchercria bancrofti. He found that 50% of D. immitis micr ifi1ariru survived fir 216 h at 5 C, 61 h at 21 C and 9 h at

37 C in saline. He fiiund that 100% mortality iccurred at 426, 352 and 48 h of cultivatiin, respectively. At

21 C 50% survived fir 74 h in distilled uator, 61 h in saline and 216 h in rabbit plasma and 254 h in plasma of uninfected dogs. He found that survival time of U. bancrofti microfilaria uas shorter than D. immitis 47

microfilaria at all temperatures and in all conditions.

Intravenous transfusion of D. immitis into dogs shouerl

microfi lariao tbo present in peripheral circulation

fir at least 50 days. Houever in rabbits, microfilariae

disappeared after 21 days. U. bancrofti microfilariae

could not be demonstrated in peripheral circulation of

rabbits or dogs, but on autopsy immodiatoly after

giv/ing infection moderate number of microfilariae were

found in lungs, livor and kidneys.

Klein and Bradley (1974) cultivated microfilariae

of D. immitis in NCTC 109 for 2-7 days and for 6-17 days

in Grace's medium supplemonted with 20% heat inactivated

horse serum. The larvae showed stomal and anal plate

far beyond those seen in L^ stage, yet no ecdysis

occurred.

riuaiko and f^kufya (ig74) used several media for

culturing in vitro, the microfilariae of Onchocnrca

V olvulus and G. qutturosa. They .ibserved the microfilariae to survive for lonqnst period if time

(8-10 and 5-6 days respectively) in distilled uat r containing 10% cattle serum and 3% glucose. Dilute

Tyrode's solution 1:1 v/v uith distilled uatcr containing 1 0/m cattle serum was found to be equally suitable. Devaney and Houells (1978) studied the development in vitn of artificially exsheathed microfilariae of Brugia pahangi in coll lines derived 48 from Aedes scutellarjs, maiayensis, A. acgypti and

Amoheles stephansj. Thoy fjund thot the 'sausage stage' icrua dcvclipod in these experiments, extrudes cells frnm anal pir.-. Thoy (ig79) studied the devclipment if Jrtifici'lly exshe-^thed microfilariae of Brugia pahangi and malayi in rrusquiti cell lines of 3 species Df Aedos and Anopheles stephensj at 28 C. ody

Deuelopment :5f micrafilari ae of B, roalayi coul^be studied in Ae des malayensis due ti the lack if available number nf parasites. Na development occurred in the absence of mosquito cells. The best results uere obtained with A. malaye nsis for both B. malayi and

B. pahangi. Up tD 13% ^f larvae reached 'sausage stage' in 8 days fir B. pahangi and up to 30^ in 6 days for

B. malayi. They (1979) carried out amther experiment using culture system for maintenance and development of microfilariae if Brugia pahangi and Dirofilaria inritis. In medium NCTC 135 supplemented with 20% heat inactivated FB5, 15-20^' if micr if i lariae developed ti late first stage larvae. Inclusion if insuct colls did not impr ive the culturt^s.

Dcvanuy and H^uells (lg79) induced the Gxshe athment

^^^ ^ Pahangi microfilariae in vitro. They isilated the microfilariae fr-^m C't bl-nd and incubatfd f-r 1 h 49

in 20 mH CaCl2 in phosphatG-froe Hank's BSS. Alternatively they UBTG incubstRd fir 30 minutes in solutions of endopoptidase (5.8 units/ml) or papaya extract protG.^se (3.0 units/ml) in calcium-free BSS. The same techniques are effective for microfilariae of

Uuchereria bancrofti and Litomosoides carinii. 50

STATEMENT OF PROBLEM

It can be seen from the foregoinq account of

historical revieu that our knowledge on various aspects

of filarial worms is quite inadequate and whatever work

has been done is meagre as well as scattered. The

morphology of different species of filarial uorns has

been studied by a number of workers only by light

microscopy. The fine cuticular structures by stereoscan

electron microscopy, have only been studied in a feu

s pedes.

The present work rjyals with a detailed

reriescrintion of Chandlere11a haukingi Chatterji, Sen

and Bhattacharya, 1965; with additional details

especially those which have been 'hne by stereoscan

electron microscopy.

The present knowledqe on the structure of

microfilariae is also incomplete. The nuclear land merks like first cell of nuslear column, nerve ring,

excretory cell, H-cell, h-cells, anal cell, last cell

of nuclear column and orqansof Schwanzqebilde are

poorly described in known poecies. The cephalic hook

and spines, the oral ring, the pharynqeal thread and

Innenkoroer are known only in a few species. 51

In the present investigation, the nuclear

structures of microfilaria of C, haukingi, have been redescribed, because in the original description, by

Chatterjee, Sen and Bhattacharya (1965), the figure does not shou a clear picture of the nuclear landmarks which are of great importance in identifying or classifying the genera of filarial worms.

Sometimes the nuclear structures are not sufficient enough for identification of genera, the cephalic structures which are speci fic for differe nt genera, have also been studied in microfilaria of

C. hawkingi.

The relative size and position of Innenkorper is also of great value in the classification of filarial worms and hence the oral ring, the pharyngeal thread and the Innenkorper have also been studied in long as well as short forms of microfilariae of C, hawkingi.

It has also been proved that the long and the short forms of microfilariae belong to C. hawkingi only, by giving ratios of body length and size of

Innenkorper, relative distance of Innenkorper, from anterior and posterior ends of the body.

The work on detailed histology of different systems of filarial worms is also limited to only two species, Brugia pahangi and Setaria cervi. The rest 52

of the uork is scattered and unsystematic. In the

follouing pages an attempt has been made to study the

detailed histology of the body wall, the musculature, the digestive and reproductive systems of C. haukingi, in vieu that certain histological characters, like musculature, the nuclear constancy of oesophagus, the cells of the intestine provide important criteria for classifying nematodes.

The uork on enzyme localization is scattered and inadequate. As the enzymes are very specific in their distribution in different genera and species, their localization provides a good taxonomic tool for separating different filariae. Investigators showed strikingly different patterns of phosphatases, comrionly acid and alkaline phosphatases in microfilariae of different genera and species. In view of this, the acid phosphatase, alkaline phosphatase, adenosine triphosphatase, carboxyl esterase and aryl sulphatase, have been studied in microfilariae of £. haukingi.

Only four of these, the acid phosphatase, alkaline phosphatase^ adenosine triphosphatase and carboxyl esterase have been studied in adult.

The Uork on in vitro cultivation has been done on a feu species only and yet not very successful.

In the present uork the in vitro cultivation of 53

microfilariae of C. haukingi has besn done as these studies are of great importance for

(a) studying the developmental stages outside

the body of the v/ector,

(b) studying the effect of various chemicals

and drugs on adult as uell as larval

stages of the parasite, and

(c) it may also help in the collection of ES

products uhich may serve as functional

antigens against filariasis, as somatic

antigens have failed to immunise the

individuals. 54

(MATERIALS AND PIETHODS

1. Olorphology

(a) Light microscopy

(i) Adult

The adult uorms uere collected from the heart in luke uarm normal saline (0.8?S NaCl) either by dissecting an anaesthetised crou or a decapitated live crou. The heart uas taken out in normal saline, dissected and examined for the uorms. The body cavity, uhich gets full of blood, was also thoroughly uashed uith normal saline and the contents uere examined for the uorms, as most of the males escape uhen the heart is cut and taken out. The uorms uere uashed thoroughly in normal saline and fixed in hot 70/S alcohol.

For studying gross morphology, the uorms uere cleared in glycerine alcohol, lactophenol or creosote.

They uere then placed on a slide under a cover-glass and manipulated by rolling them in clearing agent.

Different structures uere studied under the microscope.

The cephalic end uas decapitated and mounted in en-face \yieu in glycerine-je lly for studying cephalic papillae and amphids. 55

(ii) nicrofilaria

Nuclear structures

For studying the nuclear structures of micro- filariae, the smears uere made by taking the blood from uing uein at night. The smears uere dehaemoglobinised, dried in air and directly stained uith Leishman's stain or they uere fixed either in acetic acid-formalin- alcohol (AFA), methyl alcohol or acetone. The stains used uere either Giemsa's stain, haematoxylin-eosin,

Hallory's triple or Hallory's phospho-tungstic acid- haematoxylin.

Cephalic structures, pharyngeal thread and Innenko'rper

For studying the cephalic structures, pharyngeal thread and Innenko'rper, the smears uere made uith the blood taken from uing vein Pt night, from heparipated blood from heart or from microfilarial concentrations suspended in heparinised plasma.

Hicrofilarial concentrations uere prepared by lysing RBCs uith saponin. 5 ml of 0.5% solution of saponin in normal saline uas added to 0.5 ml of infected blood and kept at 32 C for 10 minutes. The contents uere centrifuged at 15oO rpm for 8 minutes, the supernatant uas taken out and 8 ml of Ringer's solution uas added to the sediment, thoroughly mixed and centrifuged at 1500 rpm for 10 minutes. Three such 56

washings were given by centrifuging and discarding the sunernatant. Then the sediment was suspended in heparinized plasma and smears were made, dried and fixed usually in acetic acid-formalin alcohol (AFA). Other fixatives used were ethyl alcohol 90% (warm), methanol: chloroform (2:1 m/m), Zenker's fluid or Bouin Dubosq.

The fixative was washed thoroughly in running tap water for 4-5 h. The smears were then subjected to lipid extraction by keeping the slides overnight in methanol: chloroform (2:1 v/v) at 50 C and then in absolute alcohol for several days.

After lipid extraction the smears were subjected either to oxidation or sulphation. The oxidation was done with acidified permanganate (0.3% KHnO^ with 0.2 ml conc. H^SO^ per 100 ml) or periodic acid. The latter did not give very satisfactory results. The sulphation was done by dehydrating the smears in graded ethanols, then immersing them in glacial acetic acid for 1 minute, followed by keeping in a mixture of conc. H2S0^ and glacial acetic acid (1:1 v/v) for 20 minutes at room temperature. The smears were then washed successively in glacial acetic acid, tap water and distilled water.

The oxidation was done so as to introduce the acidic group into the structures in microfilariae.

After oxidation the smears were stained with basic dyes at low pH. Aldehyde fuchsin 0.5% in 70% ethanol with 57

1.0 ml conc. HCl/10n ml; basic fuchsin 0.^% in 0.05 N HCl; crystal violet 0.1% in 0.05 N HCl; neutral red 0.5^ in

70% ethanol uith 1 ml conc. HC1/10Q ml; alcian blue in 3% acetic acid and brilliant cresyl blue Q,1/S in

0.05 N HCl.

The study of the long and short forms of micro- filariae uas done uith these preparations only.

Measurements were taken uith the help of an ocular micrometer and are in millimeters unless stated otheruise.

The drauings uere made uith the help of a Camera lucida at proper magnification. Flany details uere added in the finished drauings uhich uere inked by India ink, labelled and photographed. 58

(b) Stereoscan study

(i) Adult

The adult uorms uere taken out from the heart of anaesthetised crous. They uete kept and thoroughly washed in normal saline (0.8^). The uorms uere then kept in refrigerator at 4 C so that they become sluggish.

After about 1 h the worms uere fixed in glutaraldehyde in 0.1 n phosphate buffer and left overnight at 4 C in a refrigerator. Next morning the fixative uas washed uith 0.1 n phosphate buffer for 10-12 h uith at least

5 changes. The worms were then doubly fixed with

Caulfield's osmium tetra-oxide solution for 1 h at

4 C. Thereafter they uere washed thoroughly with distilled water and dehydrated in graded alcohols, i»e., through 30%, 5fJ%, 70%, 80%, qn|% and 95% for 10 minutes each and then twice in dry alcohol for 30 minutes each.

After dehydration was complete, the worms were passed through graded amyl acetate solution, 30%, 50%, 70%,

80%, 90%, prepared in absolute alcohol, for 10 minutes each, followed by two changes in absolute amyl acetate for 30 minutes each. Amyl acetate was used as it is miscible with both alcohol and liquid CO2, hence it is necessary to introduce it in the tissue before critical point drying with liquid C02* The worms uere then critically dried in Balzc^rs Union Type H 9202 Critical point Drier. The desired parts were mounted on stubs 59 with silver dag and dried. They uere later coated with a gold-palladium alloy in a Sputter coater

(Polaron SEH Coating Unit E 5000). The specimens ucre observed under Cambridge Stereoscan 180 Scanning

Electron riicroscope. Photographs u/ere taken an Indu

120 roll film of a speed of 125 ASA.

(ii) Microfilaria

The microfilariae were first separated from the uholG blooti. For this 50 mg phytohaemagglutinin (Difco) uas dissolved in 5 ml of distilled uater. 1 ml pf this solution uas mixed with 9 ml of distilled uater. This was used as the stock solution of phytohaemagglutinin.

For separating microfilariae 0.2 ml of this solution uas added to 0«5 ml of blood and the tube was gently rotated for 2 minutes for mixing properly the phytohaemagglutinin with the blood. To this 5 ml of

Ringer's solution uas addod and centrifuged at IDOO rpm for 3 minutes. The conglomurated RBCs, uhich become heavier uith phytohaemagglutinin, settle doun in the bottom and the clear supernatant, having microfilariae, was carefully transferred to a centrifuge tube. This supernatant was centrifuged at 1500 rpm for 10 minutes.

From this the supernatant uas discarded anci the sediment uas washed thrice with Ringer's solution by centrifuging and discarding the supernatant. Thus all the traces of phytohaemagglutinin were removed. 60

The separated microfilariae uere fixed in 4%

glutaraldehyde solution in 0.1 n phosphate buffer for

24 h at 4 C in refrigerator. They uere washed thrice

in 0.1 n phosphate buffer by contrifuging and discarding

the supernatant. They uere then doubly fixed in

Caulfield's 1% osmium tetra-oxide for 1 h at 4 C. After

osmioation the microfilariae uere uashed thoroughly in

distilled uater and dehydrated in graded ethanola, i.e., through 50^, 80%, 9^% mi for 10 minutes each and then tuice in dry alcohol for 30 minutes each, everytime by centrifuging and discarding the supernatant.

After dehydration is complete, the microfilariae uere transferred in 1:1 solution of absolute alcohol and propylene oxide. They uero resuspended in this liquid by centrifuging and discarding the supernatant. A drop of this liquid having microfilariae uas put on a cov/er- glass, dried in air and mounted on specimen stub with silver dag. These uere then coated with gold-palladium alloy in a Sputter coater (Polaron SEN coating Unit E 5000),

The observations uere made under Cambridge Stercoscan

180 Scanning electron microscope, photographs uere taken on Indu 120 roll film of a speed of 125 ASA. 61

The adult uorms uere collected in luke uarm normal saline (0,8^ NaCl) from heart oP an anaesthetised or decapitated crouj. They were straightened in hot

(60-65 C) normal saline and fixed in aqueous Bouin's or alcoholic Bouin's Dubosq fixative for a period of 14-18 h,

After fixation, they were transferred to alcohol, uhich uas also used for storage. When desired they were dehydrated, cleared and embedded in Tissu mat (55 C).

The serial sections, cut with the help of a blade attachment (AO), were flattened on albuminised slides and routine techniques uere used for staining. DPX uas used for finally mounting the sections.

The stains used uere layer's haemalum, Harris haematoxylin, Heidenhairts haematoxylin uith which iron alum 3/? uas used as mordant and 1.5^ for differentiation.

Eosin uas used for counterstaining. Other stains used are Uoigert's Iron haGmatoxylin, counter stain used being Van Cieson's picro-f uchsin, l^allory's phospho- tungstic acid haematoxylin. Picrofuchsin method of

Kattine (1962) uas also tried to stain the nuclei and nucleoli. In this method the mordant used consists of phosphotungstic and phosphomolybdic acid (1,25^ solution) in 1:1 ratio (u/v). 62

For getting batter differentiation, iron toning uos done. The sections were treated uith 1.5% after staining uith hasmatoxylin and rinsing in distilled water. This makes the nuclei black.

Mallory's triple stain was also tried. The best results uere obtained uith nayer's haemalum which u/as quite quick also. Picrofuchsin method also gave good results.

The sections were examined under microscope.

Camera lucida drawings of the sections passing through different important regions were made. The drawings were inked with India ink, labelled and photographed. 63

3. Histochemistry

(i) Adult

The adult worms uere taken out in cold normal

saline. Thoy uere washed thrice uith cold saline so as

to remove the debris present on th&ir body. The fixatior^

uas done in cold buffered formaldehyde for 18 to 24 h

at 0-4 C. The worms uere transferred to gum sucrose

and kept for 12-24 h at 0-4 C and then washed briefly

in cold distilled water. Thereafter a gelatin block was

made. The embedding was done in gelatin for 1 h at

37 C. The block was made in a small metalic cup. It was then cooled in a refrigerator, when gelatin had

solidified completely, the block was hardened in 4%

formalin for 1 h. After hardening, the block was trimmed to the desired size. It uas kept on a holder

along with a drop of water under it and allowed to

froGzc with liquid CQ2 on a carbon dioxide bench freezer.

Uhen the block has properly frozen it was fixed on a

freeze bar in cryostat. The micrometer was set at 10 yu thickness. The sections were cut by a Slec Type HS

Cryostat (Cambridge Hocking Microtome) and taken either or directly on clean slides (prechilied)/in gum sucrose solution. For picking up the sections directly on the slide, a cover slip suction pick-up was employed.

Clean cover slips or slides were picked up by squeezing the teat and expelling the air from capillary tube. The sucker disc was then applied to cov/er slip

or slide and tho teat uas released after picking up the sections by pressing the slide firmly and steadily on

the surface of the knife.

These slides unro dri^d at room temperature for

2 h. After the sections had dried and adhered properly to the surface of the slide they were incubated in the desired substrate.

(a) Acid phosphatase

Tuo methods uera used.

(1) r^odifiud lead nitrate method

(2) Coupling azo-dye method

(1) Modified load nitrate method

Sections uure incubated for one half to tuo hours

in the following solution:

2 vols 2% sodium- 5-glycerophosphate

1 vol 0.1 .^-acetfte buffer (pH 5-6)

1 vol 2.% lead acetate

0.3 vol 1-5% P1gCl2

They were then rinsed in distilled uatur and developed in ammoniacal silver nitrate solution for

30 minutes (28?^ ammonia water was added drop by drop to

5% aqueous AgNO^ untill the precipitate just dissolves) and rinsed in 5% sodium thiosulphate for 5 minutes and mounted in glycerine jelly. 65

) Coupling azo^dye method using hexazotized pararosanilin coupler

This method is quite quick and sensitive. The sections were incubated in a mixture containing 10 mg r\- naphthyl phosphate in 10 ml of acetate buffer. To uhich

750 mg of polyvinyl pyrrolidon K60 uas added and stirred uell till it became homogeneous. To this 1.0 ml of a mixture containing 0.5 ml NaN02 + 0.5 ml HPR uas added and stirred. The solution was allowed to stand before being added to the incubation mixture. The pH uas adjusted to 5. The incubation mixture having this solution uas filtered directly on to the sections and incubated at 37 C for 30 minutes. The sections uere uashed in running water for 2 minutes and mounted in glycerine jelly.

(b) Alkaline phosphatase

Two methods for alkaline phosphatase uere tried.

(1) Gomori's method

(2) Modified coupling azo-dye method

(1) Gomori's method

The sections uere incubated for one half to tuo hours in the follouing substrate containing:

10 ml of 3^0 sodium yS-glycerophosphate 10 ml of 2% Sodium di-ethyl barbiturate 5 ml distilled uater 20 ml magnesium sulphate 66

They were then rinsed in water and treated with

2% cobalt solution for 5 minutes. They uere washed in uatsr and treated with yeliou amirionium sulphide (dilute) for 15 minutes, counterstained in aqueous eosin for

5 minutes and uashed in running water and mounted in glycerine jelly.

(2) Modified coupling azo-dye method

The sections were incubated in a mixture made by dissolving 15 mg (10-20 range) sodium o(-naphthyl phosphate in 20 ml of 0.1 PI stock 'tris' buffer (pH 10). To this

20 mg of the stable diazotate of 5-chloro-O-toluidine

(salt 9 table 55 Pearse (1968) Diazonium Salts in

Enzyme Histochemistry) was added and stirred well. It was filtered directly on to the sections in a sufficient quantity to cover them at 20 C (range 17-22 C) in a

BOD incubator for 15-60 minutes. After that they were washed in running water for 1-3 minutes and counter stained in flayer's haemalum for 1-2 minutes. After this the sections were washed in running tap water for

30-60 minutes. The stained sections were mounted in glycerine jelly.

(c) Adenosine triphosphatase

The sections were subjected to incubation for 3 h in absolutely fresh medium containing: 67

0.1 n-sodium barbiturate (2.062 g/100 ml) 20 ml 0.1 Fl-CaCl^ (1.998 q/lOO ml) 10 ml Distilled water 30 ml Adenosine triphosphate (disodium salt) 152 mg (Final concentration 0.005 Pi)

pH UBS adjusted to 9.4 uith 0.1 PI >JaOH the mixture uas brought to 100 ml uith distilled uater and filtered, if turbid.

After the incubation uas complete the sections uere washed in 3 changes of 1"'' CaCl^, kept in 7.% CoCl^ for 3 minutes, uashed briefly in distilled water and deyeloped in dilute yellou ammonium sulphide, uashed thoroughly, dehydrated, cleared and mounted in UPX.

(d) CarboxylesterasG

Hnly one method i.e. HPR method was used to demonstrate carboxyIpstefase in adult worms. In this the sections were subjected to incubation in thr followinq substrate at pH 6.5.

7.5 ml of f].2 f'T phosohate buffer stock solution 2.5 ml distilled wati^r 0.25 ml substrate solution (l/J o^-naphthyl

acetate in acetone)

The mixture was gently shaken and 0.8 ml of

HPH was added. This mixture was directly filtered on to the sections and incubation was carried out for

30 minutes at 30 C. The sections were washed briefly in water, dehydrated in graded ethanols and mounted in DPX BB

(ii) nicrofllaria

Smears uicre made uith the night blood from uing

vein of heav/ily infccted crow. They uere dried in air

for 10-15 minutes, dehaemoglobinized in cold Ringer's

solution for 3-5 minutes and then fixed in buffered

sucrose formalin (pH 7.2) for 10-15 minutes at Q-4 C, rinsed in gum-sucrose solution and stored in the same

for overnight or even for longer periods. Next morning

they uere rinsed in cold saline and incubated in the

desired substrate.

(a) Acid phosphatase

Tuo methods were used.

l^lodified lead nitrate method

The smears were incubated for h in the

following solution:

2 vols 1% sodium y8-qlycerophosphate

1 vol 0.1 acetate buffer (pH 5-6)

1 vol 2% lead acetate

0.3 vol 1-5% ngCl2

They uere then rinsed in distilled uater and

developed in ammoniacal silver nitrate solution for

30 minutes (28% am-nonia uater was added drop by drop

to 5^ aqueous AgNO^ untill the precipitate just dissolves-)

and rinsed in 5% sodium thiosulphate for 5 minutes and

mounted in glycerine jelly. m

(2) Coupling azo-dye method using hexazotized pararosanilin coupler

The fixed smears of microfilariae were incubated

in a mixture containing 10 mg c<-naphthyl phosphate in

10 ml of acetate buffer. To uhich 750 mg of polyvinyl

pyrrolidon K60 was added and stirred uell till it became

homogeneous. To this l.n ml of a mixture containing

0.5 ml NaN02 (4%) + 0.5 ml HPR uas added and stirred.

This mixture uas allowed to stand before being added to

the incubation mixture. The pH uas adjusted to 5. The

incubation mixture having this solution uas filtered

directly on to the smears and incubated for 30 minutes

at 37 C. The smears were uashed in running uater for

2 minutes and mounted in glycerine jelly.

phosphatase

Only one method i.e., modified coupling azo-dye

method uas used to demonstrate the alkaline phosphatase.

The smears uere incubated in a mixture made by

dissolving 15 mg (10-20 range) sodium o^-naphthyl

phosphate in 20 ml of 0.1 H stock 'tris^ buffer (pH 10).

To this 20 mg of the stable diazotate of 5-chloro-G-

toluidine (salt 9 Table 55 Pearse (1968) Diazomium

salts in Enzyme Histochemistry) uas added and stirred uell. It uas filtered directly on to the smears in a

sufficient quantity to cover them at 20 C in a BOD 70 incubator for 15-60 minutes. After that they uere washed in running water for 1-3 minutes and mounted in glycerine jelly.

(c) Adenosine triphosphatase

The smears uure subjected to incubation for 3 h at 37 C in absolutely fresh medium containing 20 ml of

0,1 n-sodium barbiturate (2.062 g/100 ml); 10 ml of

0.18 n CaCl^ (1 . 998 g/100 ml); 30 ml of distilled water and 152 mg of adenosine triphosphate (disodium salt).

As soon as ATP was dissolved, the pH was adjusted to

9.4 with 0.1 n NaOH and the mixture was brought to

100 ml with distilled water. It was filtered, if turbid.

After the incubation is complete the smears wore washed in 3 changes of CaCl2, transferred to 2% C0CI2 for 3 minutes, developed in dilute yellow ammonium sulphide, washed thoroughly, dehydrated, cleared and mounted in DPX.

(d) Carboxylesterase

The smears were incubated at 37 C for 3 h in a mixture prepared by dissolving 1,3 mg of 5-bromoindoxy1 acetate in 0.1 ml of absolute alcohol and adding to it

2.0 ml of 0.1 M tris-HCl buffer (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 ferrocyanidc

1.0 ml of 0.1 n CaCl2 and the

volume is made up to 10 ml uith distilled uater.

After incubation the smears uere rinsed in uater, deihydrated through ethanol grades and cleared in 1 part phunol 3 parts xylune and mounted in DPX.

Another method used for carboxyl esterase uas the HPR method. In this the smears uere subjected to incubation in the following substrate at pH 6.5.

7.5 ml of 0.2 PI phosphate buffer stock solution

2.5 ml distilled uater

0.25 ml substrate solution (1% c/-naphthyl acetate

in acetone).

The mixture uas gently shaken and 0.8 ml of HPR uas added.

This mixture was directly filtered on to the smears and the incubation uas carried out for 30 minutes at 30 C. The smears uere uashed briefly in uater, dehydrateri in graded ethanols and mounted in DPX.

(e) Aryl sulphatase

Fixed smears uere incubated for 1 h in freshly prepared mixture of 72

^ -hydroxyquinoline sulphate 50 mg

[\laCl 200 mg

l/cronal acetate buffer pH 6.0 10 ml

Hexazonium pararosanilin (fresh) 0,6 ml

pH 5.8-6.0 (adjusted uith NaOH)

The smears were uashcd in distilled uatcr, dehydrated, cleared and mounted in DPX. 4. I_n \/itro cultivation

Only microfilariae uere cultivated ^ vitro.

Night blood uas taken from the uing vein of heavily

infected jungle crows. Heparin was added to this blood

(10 units/ml) to prevent co-agulstion. Whenever desired

the infected crow was sacrificed, during day, the lungs

were taken out and kept in warm (37 C) heparinized

Ringer's solution for sometime and microfilariae were

taken out from the lungs by teasing them gently. After

15-20 minutes the pieces of lungs were discarded and

microfilariae were separated from this Ringer's solution

containing lung exudate. The microfilariae could also

be obtained in large numbers from the blood directly

taken from heart with the help of a syringe.

Before carrying out vitro cultivation the

concentrated yields of microfilariae were obtained by

separating them from blood corpuscles. This could be

done by;

1. Conglomerating RBCs

2. Lysing RBCs

Different reagents were used for obtaining the microfilariae from the blood.

1. Phytohaemagglutinin

Liith phytohaemagglutinin the RBCs get conglomerated

and settle down quickly leaving microfilariae free in 74

plasma. 50 mg phytohaemagglutinin (Difco) was dissolved

in 5 ml of distilled uater. 1 ml of this solution

uas mixed with 9 ml of distilled uater. This uas used

as a stock solution. 0.2 ml of this solution uas added

to 0.5 ml of blood and the tube uas gently rotated for

2 minutes for mixing properly the phytohaemagglutinin uith the blood. To this tube 5 ml of Ringer's solution

uas added and centrifuged at 1000 rpm for 3 minutes.

The RBCs settled down in the bottom and the supernatant

containing microfilariae was carefully transferred to

a centrifuge tube and centrifuged at 1500 rpm for

10 minutes. The supernatant uas discarded and the

sediment uas uashed thrice uith Ringer's solution by

centrifuging and discarding the supernatant. Thus all

the traces of phytohaemagglutinin were removed. The

separated microfilariae uere examined under the microscope for their viability. This uas supposed to be the best method as it gave maximum yield and the visbility of microfilariae uas not affected.

Uith phytohaemagglutinin, the RBCs get conglomerated

and so they settle doun quickly leaving microfilariae frne in supernatant. The purpose of adding more Ringer's solution immediately after addition of phytohaemagglutinin uas to prevent settling doun of microfilariae which would have normally happened if the volume was less. 75

2. Uater

In this method 1P ml of distilled water was added

to 0.4 ml of infected blood. It uias mixed by shaking

gently and uas allowed to stand for 2 minutes and

centrifuged at 1500 rpm for 8-10 minutes. The

suoernatant was discarded. The sediment was mixed with

Ringer's solution and washed twice by centrifuging and

discarding the supernatant. This method did not give

very good yield.

3. Saponin

For lysing RBCs, saponin was found to be very effective with subsequent treatment with trypsin for

getting rid of the remaining cellular constituents.

In this method G.5% solution of saponin and 0.1% solution of trypsin were prepared in normal saline and

kept in water bath at 36 C. 4.5 ml solution of saponin was added to 0.5 ml infected blood and kept at 32 C

for 10 mihutes. The contents wore centrifuged at 1500 rpm

for a minutes. The supernatant was discarded and to the sediment 8 ml of Ringer's solution was added,

thoroughly mixed and centrifuged at 1500 rpm for

10 minutes. Three such washings were given by contrifuging everytime at 1500 rpm for 10 minutes and discarding

the supernatant. After the final wash 4 ml of 0.1% trypsin solution in normal saline was added to the sediment, mixed well, incubated for 5 minutes at room 76

temperature and centrifuged at 1500 rpm for 10 minutes.

The supernatant uas pipetted out and the sediment uas

uashed thricc with Rinq.'.r's solution.

Somotimes first treatment failed to lyso all

the RBCs in uhich ease a second treatment with Q.2%

saponin solution uas given for 2 minutes.

Saponin uas found to be very effective uith

subsequent trf.atment uith trypsin for getting rid of

remaining cellular constituents. This method gave

good yield of microfilariae but saponin is found to be

toxic to the microfilariae. Since the viability of microfilariae is affected, this method did not prove

to be good for ^ vitro culture as the microfilariae

did not survive even for normal periods of time.

While using this method another problem arose

due to the nucleated RBCs of crou. After treatment

with saponin uhen the blood uas centrifuged the microfilariae uere found to be entangled in a mass

having nuclei of RBC. Trypsin could not remove this

complete ly.

A number of experiments uere performed to

cultivate ^ vitro the microfilariae of Chandlere11a hsukingi and induce their development. (*1any salt

solutions, chemically defined media and biological

supplements uere used. The microfilariae uere

separated from blood and concrntrated. This concentratcri 77

yield of microfilariae uas then dispensed into a series of culture vials having different media uith antibiotics comprising 100 units p^'nicillin and SOyug streptomycin.

The culture tubes uere then incubated at 22 C in a BOD incubator.

All glassware were heat sterilised. The basic salt solutions uore sterilized at 15 lb pressuru in an autoclave. The biological supplements viz., serum, CEE and defined media NCTC 109, Eagle's PIEM, Medium 199,

Cr-IRL 1066 and Grace's insect tissue culture medium GP1A uere sterilized by filteration in Seitz or Plillipore filters. The serum u?s inactivated at 56 C for 1 h in a water b^th before use. The experiments uere carried out under axenic conditions. The media uere not changed throughout the culture period as there uas every possibility of mort'^.lity and damage to the microfilariae uhile centrifuging and washing due tn their delicrte structure. P number of media and supplements uere usud for in vitro cultivation. 78

RESULTS AND DISCUSSION

1. (Morphology

(a) Light microscopy

(i) Adult

Chandlerella haukingi Chatterjee, Sen and

Bhsttacharya, 1965.

The body is elongated uith attenuated ends. The cuticle appears smooth and devoid of striations under the light microscope. The mouth is terminal, slit-like without lips. There are tuo pairs of cephalic papillae, one pair sub-uentral and the other sub-dorsal. A pair of amphids are present, one on each lateral side.

Plale

The male is smaller than the female, 11.85-14.21 mm in length and 0,175-0,212 mm in width. The cephalic end is bluntly pointed. The nerue ring is present at

0.137-0.187 mm from the anterior end of the body and around muscular part of oesophagus. The excretory pore is present 0.112-0,125 mm from the anterior end of the body.

The posterior extremity is blunt and curved making tuo to three coils. There are five pairs of post-cloacal papillae. The spicules are sub-equal and dissimilar uith truncated proximal ends and rounded tips.

The left spicule measures 0.059-0, 072 mm and the right

0.048-0.062 mm in length. 79

The mouth liBads into the oesophagus uhich is

divisible into tuo parts: a short anterior muscular

part and a long posterior glandular part. The former

measures 0.125-0,212 mm uhile the latter 0.46-0.61 mm.

The tuo parts together measure 0. 59-0. 82 mm in length.

The intestine runs in a straight course and continues

into rectum. The cloacal aperture is present at

0.175-0.18 mm from the tip of tail.

The testis is convoluted occupying approximately tuo thirds of the body from posterior end, extending

anteriorly up to the middle of the glandular part of oesophagus. The uas deferens is coiled, followed by vesicula seminalis which gradually widens and finally narrows down forming ejaculatory duct which opens into the rectum forming common cloaca.

Female

The female is longer than the male, 15.86-21.19 mm in length and 0.24-0.31 mm in width. Both the extremities of body are bluntly pointed. The nerve ring is present around muscular portion of oesophagus, at 0.12-0.15 mm from the anterior end of body. The excretory pore is located at 0.125-0.20 mm from anterior end of the body.

The mouth opens into the oesophagus which is divisible into two parts, the muscular part 0.125-0.275 mm and the glandular part 0.4-0.44 mm, the two parts 80

together measure 0.5-0.55 mm. The intestine does not open to the outside. The anal aperture is atrophied and represented by a depression, at 0.35-0,37 mm from the posterior end.

The ovaries are parallel, opisthodeIphic; the tuio uteri run side by side in a convoluted uay and unite to form a common vagina. The v/ulva is present at 0.30-0.50 mm from the anterior end of body, l/iviparous, the uterus is full of developed microfilariae. B1

(i i) nicrofilarja

Nuclear structures

The microfilariae measuring 0.142~0.164 by n,005 mrn are couered by a delicate sheath uhich has been stained by 3 1C i 3 n blus at pH 5.0. The body is full of nuclei but a small anterior part devoid of nuclei, is known as cephalic snoce, r^easurinq

0.004-0.005 mm in length. The nerue ring is present at 0.04-0.05 mm and the excretory pore at 0.05-0.06 mm from anterior end of the body.

The posterior end of microfilaria contains a series of 4 cells and anal vesicle. The first is G^ cell having large vacuolated nucleus with nucleolus, present at 0.042-0.044 mm from Dosterior end of the body and the rest are R^ , R.^ and R^ cells.

The anal vesicle is seen behind H-cells, present at 0.016-0.01R mm frnm posterior end of the body. The paired organs seen betuieen thn tip of tail and anal vesicle are known as Schuanznebi Ide ui'th separate ducts opening outside, situated at n.n0'3-0.nn4 mm from posterii:)r end of the body. These are pernaps sensory in function. The nuclear column continues up to the tip of tail and there is a sinale nucleus near the tip fFig. 1). 82

Cephalic structures, oharynqeal thread and Innenkorper

The cephalic structures in the microfilaria of

Chandlerella haukinqi have been studied with a vieu

that they are characteristic of the genera of filarial

uorms and this uould help in identification of different

genera from microfilariae alone.

Results

'3f the dyes used after oxidation, aldehyde

fuchsin gave the best results. Liith this stain, at the

tip of cephalic space, a hook was discernible uhich is

curved at its distal end and bifurcated at the base.

iMear the hook there are four small tooth like structures,

the spines, arranged in a transverse rou uhich look

like equi lat'-^'r al traingles (Fiq. ?). There is also

a uell defined ring like structure - the oral ring.

From near the centre of this ring the oharynqeal

thread starts as a funnel shaped structure. It runs

internetlly and rentrally to end in the Innenkoroor uhich

is situated in the antfri ir nart of the posterior third

of the body of the microfilaria. The Innenkorper is

rouqhly rectangular in shape. The cuticle and the

sheath could also be defined very clearly. The sheath

looks like a smooth covering around the body of the microfilaria. The space betueen the cuticle and the

3 he ath remains almost uniform throughout the body 83

length except at the posterior end uhere it is stretched apart. In this repion, tetueen the cuticle and sheath, some stained objects could be seen. The cuticle is transversely striated all over the body and at the posterior end thickening of a Feu cuticular striations is observ/ed. A spine is seen jn the cuticle at the tip of the posterior end ''Figs. 3 I 4).

Other dyes like basic fuchsin, crystal violet, brilliant cresyl blue, neutral red and alcian blue stained the hook and spines equally well but none of them gave a clear differentiation of the pharyngeal thread. The Innenkorper could be stained well with basic fuchsin, crystal violet and alcian blue.

Aldehyde fuchsin also gave fairly good results after sulphation but the structures present at the anterior end of the cephalic space, i.e., the hook, spines and thn oral ring could not be distinquished

SGparatf ly. The tip nf the cephalic space took fairly qood stnin uith othor dyes like basic fuchsin, crystal violet and brilliant cresyl blue. After sulphation pharynncal thread could be stained only uith aldehyde fuchsin. Innenkorper urns intensely stained uith aldehyde fuchsin, basic fuchsin, crystal violet, bri lliant rresy 1 blut^, ntutral rod and alcian blue.

Sheath could be stained uell uith alcian blue and basic fuchsin but not uith other dyes. Cuticle could 84

be defined uell only when stained uith aldehyde fuchsin

and basic fuchsin. Comparative efficacy of different

stains after oxidation and sulphaMon has beon

summarised in Table 1.

Discussion

The staining roactions of the micnfiJariae of

Chandlerella haukinoi are quite similar to those of

Brugia (Laurence and Simpson, 1969) uith some

differences. After ox id at ion, aldehyde fuchsin q?\yo

almost equally good results in both ncratodns in

differentiating the structures in the cephalic space

the Innenkorper, the pharyngeal thread, cuticle and

sheath. With basic fuchsin fair staining of the

cuticle and sheath could be achieved in case of microfilaria of Chandlerella haukinqi but not uith microfilaria of Brugia, 'Staining reaction to crystal

v/iolet ujas similar in both the species, though uaryinn

in intensity. Uith brilliant cresyl blue, neutral red

and alcian blue thpre uas not -^uch difference butucjen

the tuo species.

After sulphation also, aldehyde fuchsin gave

the best results. It stained the cuticle of microfilaria

of Chandlerella haukinqi mirn intensely than that Tf

Erugia. But uith basic fuchsin results were not as ttD

qood as observed by Laurence and Simpson (1969) in

Bruqia. Uith crystal violet, Laurence and Simpson

(1969) reported intense staining of nharyngeal thread and poor staining of cephalic structures. But it has been just the opposite in ease of Chandlrella.

Laurence and Simpson (1Q69) reported intense stainino of cephalic structures and pharyngeal thread with brilliant cresyl blue but it uas not so obvious uith

Chandlerella. Hinor dissimilarities were also observed with neutral red and alcian blue. According to oresent evaluation of the stains studied, aldehyde fuchsin after oxidation seems to be the best for routine examination of microfilariae in smears.

Usino the different staining techniques, it has been possible to study in the microfilaria of Chandlerella haukinqi some important structures uhich sbp'v to differ, from snecies to species, in their charc-'ctor and arrangement. Table 2 gives a compamtive summary of the distinquishing characters of these structures in microfilaria of different species of filariae so far studied by Laurence and Simpson (1169). Fasud on this a key to a tintative classification of filariae is appended belou. Using ^he key it is honed that i-he common species of filariae could be identified, in the absence of adult uorns, from microfilariae alonn and uould be useful in diagnosis esoeCially in cases of zoonosis. 86

Key to the genera of filarial uorms based upon cephalic structures

1. Hook bifurcate at the base uith spines 2

Hook not bifurcate, spines absent 5

2. Hook uith transverse supporting bars 3

Hook without supporting bars A

3* Hook U-shaped

Spines 4, triangular Uuchereri a

Spines 3, triangular Brugia patei

Spines 3, fang-like B, malayi and

B. pahangi

Spines numerous, arranged irregularly in 2-3 rous Loa

4. Hook fang-like, spines 4, triangular in shape Chandlerc11a

5. Hook uith tuo transverse supporting bars 6

Hook without transverse bars 7

6. Hook bou-shaped Cardiofilaria

7. Hook broad plate like uith central Onchocerca posterior projection ^lansone 11a Hook small tooth like structure f

Hook still smaller Dipetolonema

Hook beak like Litomosoides 87

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Fin. 2. ucphalir 3tructij"^os jF micrDfi 1 aria, 92

E E

Fig. 1

h h -s o V fP /

o V

fig. 2 Fiq. 3. ]ral ring, nharynqeal thread, Innenkorper

and tail spine in long Trm of

micnf i laria.

Fin. "ja-c 3tructurrjs in shnrt form of

n i L- n f i 1 r, I i a. 93

(^ig. 3

Tig. 4 94

Forms of microfilarias

Uhile studying the smears for microfilariae, tuo

forma of microfilariae uare observ/ed in C, haukingi.

The long and the short forms^ Both tha forma showed identical features. The sheath, the cuticle, the hook and spines, oral ring, pharyngeal thread and Innenkorper uere all identical. The extent of pharyngeal thread, the position, shape and size of Innenkorper is similar in relation to the body size. The long forms of microfilariae, measuring 136.4-174.9 jj, 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 microfilariae, a feu intermediate forms

are also available. The size of Innenkorper, the storehouse of mucopolysaccharides, also decreases uith the shortening of the body(rigs. 5 & 6 and Tables 3 & 4).

Further, to verify that these tuo forms belong to one species, a number of short, intermediate and

long f':irms have been measured, the position of

Innenkorper has been noted and the tuo ratios, between the body length and distance of Innenkorper from anterior end as well as between the body length and distance of Innenkorper from posterior end, have been uorked out. It is found that these ratios 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 microfilariae show constant oosition of

Innenkorper. The nuclear landmarks and the cephalic structures are similar in both long and short forms.

These observations go to prove that the size of microfilariae decreases uith the decrease in size of

Innenkorper - the storehouse of mucopolysaccharides, due to the immune responses of host. As the infection gets older, the concentration of antigens increases in the blood and the microfilariae do not get proper food from their surroundings. Their size decreases uith the decrease in size of Innenkorper as microfilariae use up the stored glycogen from it for their survival. 96

Table Length of microfilariae, size and position of" Innonkorper.

S. Len th f .Size of ,Oistanco of .-Microfilaria . T nnenkomer ,Innenkorper •Lennth'Breadth'anterior

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 nn.o ^.3 3.3 81 .4

21 11 2.2 3. ^ 84.7

contH. on next page Table 3 ' contri.)

' ( S.N. Length of t J i z e o f tPistanc s of '"'iicrofilaria ilnncnkorpar t Innenkorper 'Lenqth'Brparth, from antt^rior I t end 1 > 1

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 88

Table 3 (contd. )

I t , 3 i2e of ,Distance of S.N. , Length of JInnenkorper , Innenkorper f^icrof i laria I Lfjngfeh i Bre adth (from anterior 1 I t , 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 99

T ah 1G 3 (cantd.)

,3ize of ,Distance of 3. N. , Length of ,Innenkorper ,Inne nkorpe r , l^icrof i laria ,frnm anterior 'Length'Breadth T f , 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 100

Tablf-' ''contd. )

t3izr' of .Distance of S.N. _ L&ngth of .Innonknrpor .Innenkorper Nicrofilaria 'Length 'Breadth , from anterior ! 1 ,and

86 1 62. B 3.3 110. 0

37 1 63. 9 4.4 4.4 114.4

88 169.4 3.3 4.4 112.2

89 1 69.4 4.4 4.4 122. 1

90 170.5 3.3 3.3 11 6. 6

91 174. 9 3.3 3.3 114.4

All mGnsurcments are in p 101

Table 4. Decrease in the size of Innenkorper uith the shnrtening of the body.

j.!\l. Body lenqth Size 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

All measurements are in P- 102

E o (-1 to LD cn O • • • • • u CO LO UD 01 CO CN CM CM CM OJ a o u o -p ^ CO CM c n CD r- ID CO o c • • « • • c >X3 tn LO Ln J—( t- r- r- E P o U CD ci_ o C c O- f-i en CD n CD -P Q f-1 tn QUO •H o -.n -w •o c ^ p m o m fi CM a\ CO c Q} to CM CM CM •a • -4-) Q) -P c •D cn c CO ro C •H c o 0 C) o x: •p (H cn O c •H (D e -H CD n -P M cn Ct- n o •o o Q- 'I- p c X3 o a a cn a. CD CO 0) f-i PI JZ O r CM cr o CO ro -P r-i c cn v£) lT) c r-H CO C M c CD CD D CD 3 cn c 4J (D CD c c U 3 CO O t-H CD CO -P CO CD CD XI M -P n -C C cn •M -P cu n* (-4 £ •tH CD c 03 4-> -P CD U CO C D u:: CO CJI CM m in CM cn CM O cn CO r- CO X] Q o E LO CD (D JD 'I

oi to LD rn Fig. S. Photomicronraph of long form of

microfilaria. X 1100. 103

»1 Fig. 6. Photomicrograph of short form of

micnPilaria. X noo. 104

Fig. 6 105

(b) Stereoscan sturiies

(i) Adult

The body cuticlc has bpRO doscribod to bt. Gmooth

by light microscopy but it shous strong striations at

anterior and postorior ends of body. The mid rngion

of body shows feeble striations. The mouth is elliptical, mounted on a raised shield of cuticlc,

Nothing special could be observed regarding copha lie

oapilloe and amphids, only that amphids are su^;n to

have a peduncle. The anal depression in female is

found to be surrounded by feeble uauy striations uith

a feu small uarts. Wo difference was observed in

cephalic structures of Tiale and female. The tail in

female shous striations uith bluntly rounded tin. The

tail of male dot_3 not show any additional features

(Figs. 7-13).

(ii) Hicrofilariao

The stereoscan studies of microfilariae did not reueal anything soecial as hhey are cov/erud uith a sheath only; the sheath shows wrinkles at th(i posttrior end in long forms and it is wrinkled throughout in short forms (Figs. 14 & 15). Fig. 7. Scanning elcctnn micrograph of anterior

pnd of adult. X 1500.

Fig. 8, Scanning rlectnn micrograph of head

en-face uieu of adult. X 1000. Fig. 7

Fig. 8 Fig. 9. Scanning electron micrograph of general

cuticular surfacFj in midbody reginn of

adult. X 5000.

Fig. 10. Scanning electron micrograph of vulva

of female. X 700. 107

Fig. 9 /ig. 11. Scanning electron mirrnqraph of cuticular

ornamentation around anal depression

of female. X IIOOG.

Fig. 12. Scanning electron micrograph of tail

of female. X 108

Fig. 11

Fig. 12 Fig. 13, Scanninq electron minrnqraph of tail

of male. X 109

Fig. 13 Fig. 14. ocannin-. electron micrograph of short

form of microfilaria. X 3100.

Fig. 15. Scannin-'' electron rriicronranh of anterior

find of short firr of microfilaria, X 11000. 134

Fig. 14

Fig. 110 Ill

2. Histology

The present uork deals uith the histological studies of Chandlere11a haukingi, a filarial parasite of Indian jungle cnu Cprvus macrprhynchos (Uagler).

BODY IxJALL

The body uall consists of cuticle, sub-cuticle and muscle layer. These layers are very intimately associated uith each other.

Cuticle

This is outermost, semitransparent covering of the body. Besides covering the external parts it passes through all apertures like mouth, vulva and cloaca, forming internal lining of oesophagus, vagina and rectum,

The superficial layer of cuticle forms papillae, cephalic as uell as post-cloacal and striations on the body (The cuticle shous strong striations in anterior and posterior part of the body and feeble striations in the middle region of the body by stereoscan electron microscopy). In transverse section the cuticle shows five layers (Fig. 15 ). These layers are as follows:

1. Outer cortical layer 2. Fibrillar layer 3. Matrix layer 4. Fibrous layer 5. Basement membrane 112

The external cortical layer is quits dense as compared to the other layers. Matrix layer appears like a spongy layer. The fibrillar layer is perhaps formed by condensation of matrix layer. In this layer the

fibrils form the netuork. The fibrous layer is made up of connective tissue. The basement membrane is a thin layer.

These layers arc not present throughout the body. Only outer cortical layer is present in the lining of oesophagus v/agina and cloaca. The cuticlc is thicker in male than in female, especially in the greater part of the middle region of body. This may be due to the enormous development of uteri in female.

Discussion

The cuticle of the present form resembles the cuticle 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 cervi. Goldschmidt (1906) described

9 layers in the cuticle of Ascaris lumbricoides.

The nature of cuticle has also been discussed by some uorkers. Grubbe (1850) termed it chitin.

Flury (1912) called it keratin and Plagath (1919) termed it cuticle in case of Ascaris. Mueller (1929) described the cuticle as a secretion product, but did not mention the nature of the product. Chituood (1936) 113

and Ansari and Basir (1964) bsliewed it to be proto- plasmic condensati-TH from living cells. The author also agrees with the vieu of Chj.tunod (1 936) and Anaari and Basir (1964).

Sub-cuticle

It is present belou the cuticular layer in the form of 3 fine sheet of protoplasm. It becomes thin towards both the extremities but becomes thicker in the middle region of the body in male and in posterior part of the anterior one third of the body in female.

In the middle region of the body of female, the sub-cuticle gets pressed due to the enormous grouth of uteri. This layer bulges into pseudocoelom at four places in the form of longitudinal chords; one dorsal, one ventral and tuo laterals, one on either side.

The dorsal chord is thinner than the ventral uhile the laterals are most prominent. These chords divide the musculature into four groups. The nuclei are seen only in the chordal region. All the four chords contain nerve cells and the lateral ones lodge lateral excretory channels also. The chords begin as small thickened structures in the anterior region of the body where nuclei are not seen in it. These chords become modified at different levels. In oesophageal region they become high giving support to the oesophagus by surrounding it partly (Fig. 17), These give support 114

to vulva in female. In the region of gonads the chords

become highly flattened. In tail region they become

inconspicuous.

Discussion

This form resembles B. oahangi, as described by

Schacher (1962), in having four sub-cuticular chords.

Ansari and Basir (1964) described four additional ridges, tuo sub-dorsal and two sub-ventral lying on either side of lateral chords in case of cervi.

Hany workers have discussed its origin also.

Hamann (1895) regarded it ectodermal while zur Strassen

(1904) believed it to be mesodermal in origin. Stewart

(1906) called sub-cuticle as hypodermis. Schacher

(1962) did not discuss its origin.

flUSCULATiJRE

Inside the sub-cuticle lies the somatic musculature. The four sub-cuticular longitudinal chords divide the musculature into four sectors. The somatic musculature gets modified forming specialised muscles in oesophageal, intestinal, vulvar, anal and spicular region.

The somatic musculature is coelomyarian, polymyarian type, each muscle cell consists of a proximal fibrillar part and distal protoplasmic nucleated part. The fibrillar part is attached to the 115

sub-cuticle and protoplasmic part lies Free in the pseudocoel (Fig. The supporting fibrils are present in orotoplasmic part. These fibrils have connections uith the nervss running inside the chords.

The specialised muscles supporting oesophagus are knoun as aomato-oesophageal muscles. They extend from somatic musculature to oesophagus to giv/e support to it. These muscles are attached to the oesophagus at the lev/el of or a little behind the nerue ring. These muscles do not form any definite pattern (Figs. 17 ^ 19).

Those supporting the intestine are termt-a .'s somato-intestinal muscles. These are in the form of bands or a sphincter around the intestine (Fig. 20). In male the spicules are supported with protractor and retractor muscles. In female, these muscles give support to uulua and are knoun as somato-vulvar muscles.

Discussi m

Uhile studying the musculature of nematodes

Schneider (1B60) found that the form, number and arrangement of muscles are of great importance in classification of nematodes. On this basis he grouped the nematodes into tuo groups, platymyarian and coelomyarian. The platymyarian are the nematodes in uhich the fibrillar portion of muscles is flat towards the body cavity and coelomyarian are those in uhich the fibrillar portion of muscles bear groove and the 116

protoplasmic part dip down into the body cavity

(pseudocoel). Further on the basis of the number of muscle cells in each scctor, he suggested tuo groups of nematodes, mernmyarian hau/ing feu muscle colls in cach sector and polymyarian, if they are numerous,

Hartini (1916) restudied the musculature of nematodes and suggested that polymyarian and coelomyarian nematodes are raeromyarian and platymyarian by finding the transitional forms between the two in the larval stages.

Schachei' (1 962) did not discuss this point in the case of Brugia pahangi.

Ansari and Basir (1964) suggested that polymyary and coelomyary is reached in later stage of devolnprnent.

They described somatic musculature in Setaria cervi as polymyarian and coelomyarian type. They also described the specialized muscles, the somato-oesophagcal muscles, somato-intestinal muscles, somato-uulvar muscles, somato- rectul muscles, musculus ani, copulatory muscles and spicular muscles.

The musculature in Chandlerc11a hawkingi is almost similar to the musculature of ceryj only with few minor differences. In the former musculus ani and copulatory muscles are not observed. 117

BODY CAUITY

The body cavity is a pseudocoel, anteriorly in

the region if muscular oesnphagus and posteriorly in

tail region, the body cav/ity is completely obliterated

due to the enormous growth of connective tissue. In

female, in the middle region of the body almost all the

space is filled with coiled reproductive organs. The

body fluid is granular. Some nucleated connective tissue

is also seen in pseudocoel in oesophageal region.

Pi s cus si on

Goldschmidt (1906) described the body cavity in

Ascaris t.o be surrounded by 'isolation tissue' uhich is

a membranous tissue supporting various organs. Steuart

(1906) found nuclei in jelly like mass in Oncholaimus

and believed the body cavity to be filled uith

mesenchyme but because hn could not see the cell uall

around the nuclei he did not emphasize his opinion.

Ansari and Basir (1964) gave different views regarding

the body fluid. They suoqested that the body fluid is

either a fluid uhich gets co-agulated when the worms

are treatbd with fixative or it is a mesenchymatous tissue of lou organisation. They also presumed it to be

a fluid in place of circulatory fluid.

^^ £• haukingi the body fluid is jelly like

granular substance uith some nuclei in it. It may be a

fluid in place of circulatory fluid. 118

CQMNECTI\JE TISSUE

It is nucleated, granular mass of tissue present

around the oesophagus. It is not seen in any other region in this case.

Discussion

The connective tissue uas given different names.

Schneider (19Q2) called it " Bindegeuebe" . Lojss (1905) named it "strand like organs". Goldschmidt (1906) proposed the term "isolationgeuebe" for it. Ansari and

Basir (1964) suggested it to be like a mesentry holding various internal organs in their place. In Chandlere11a hawkingi this tissue is like a mesentry and limited only to oesophageal region.

DIGESTIVE SYSTEM

Digestive system consists of mouth, oesophagus, intestine, rectum, opening out through cloacal aperture in male and ending blindly in anal depression in ftjmale.

The mouth is simple, present on raised cuticular plate. It leads into oesophagus.

Oesophagus

The oesophagus is divisible into two parts, the anterior muscular and the posterior glandular. It is covered by a thin membrane, the tunica propria. 119

The muscular part of oesTphagus is smaller and narrouer than the glandular part of oesophagus. Its ualls are thick^ muscular uith a triradiate lumen. The lumen is supposed to be formed of cuticle uhich covers the b3dy of nematode externally. The glandular part of oesophagus is lonqt^r and broader than the muscular part

if oesophagus. Its ualls are syncytial. This part often shows slightly muscular charactor around the lumen, frim uhich muscular rays are seen projecting into syncytial mass. The lumen is often circular. The muscular part of oesophagus is supported by somato- oesaphageal muscles uhich start from body uall musculature to give support to oesophagus (Figs, 17 & 19).

The glandular part of oesophagus is supported by sphincter muscles (Fig. 21 ).

A nerve ring is seen around the muscular part of oesophagus. It is present obliquely as in sections it is always Sfjen in semilunar form (Fig. 22). Hugo quantity of connective tissue is found to fill up the space between the body wall and the oesophagus (Fig. 23).

Intestine

The oesophagus is followed by intestine which is thin walled. The ualls are made up of a number of cells and hence it is of myriocytous typs showing 6-10 nuclei per section. The cells of the wall are of epithelial type (Fig. 20 ). Re ctum

The intestine becnmes slightly wider forming

rectum uhich ends blindly in an anal depressiin in

female and opens out through cloaca in male. Rectum is

lined internally by a cuticle which is a continuation

of the external cuticle,

Cloaca

Cloaca is a common cavity formed by recturm,

and ejaculatory duct opening into it. It is lined internally by cuticle. Spicules are often seen projecting through this opening(Fig. 27).

Discussion

The nature of oesophagus of C. haukingi is

similar to the oesophagus ]f B_. pahanqi as described by

Schacher (1962) and 3. cervi Ansari and Basir (1964).

The only difference is that the glandular part of

oesophagus -'ften shows a circular lumen instead of a triangular one.

The intestine is also of myriocytous and

anisocytous type as categorised by Chituood (1930).

The walls hav/e large number of cells and the lumen is irregular. pahangi and S. cervi also fall in the same category. 121

mil REPRODUCTII/E SYSTEM

The male reproductive system is tubular and rmmrchic. The testis is thread like and coiled. It begins uith ane cell in the muscular part nf oesophagus and proceeds backuards and becomos several cell thick.

The testis leads into tubular vas deferens of approximately same width as that of testis. The vas deferens is quite long and leads into uider seminal vesicle which is followed by muscular ejaculatnry duct.

The ejaculatory duct opens into rectum just in front of the cloacal aperture. This common tube is known as the cloaca.

Testis

The testis consists of two parts, the germinal zone and the growth zone. The germinal zone starts with one cell but the number increases gradually as it proceeds posteriorly. This z:no is covered over by flat cells. The g.^rm cells inside this covering are compact (Fig. ).

The germinal zone passes insensibly into growth zone. This zone is also coverea over by flat cells.

The cell lining is thin in the anterior region but becomes thick posteriorly. The cells inside this covering are known as spermatogonia (Fig. 2lf ). 2

l/as deferens

The gnuth z )ne is f-^llnuod by vas deferens uhich is also nf the same width. The uslla are glandular and the lumen is full if dcuclopinQ spermatoz:)a uhich are small spherical structures (Fig. 25).

Seminal vesicle

The uas deferens uidens to form the seminal vesicle. This part stores the sperms. The ualls are made up of flat and narrou epithelial cells. The lumbn is full of fully developed spermatozoa (Fig. 26 ).

Ejaculatory duct

The seminal vesiclQ is folloujod by muscular ejaculatory duct. The outer layer of ualls is of circular muscles and the inner layer is of columnar cells. This duct opens intD common cloaca (Fig. 27).

Spicules

The spicules are sub-equal, dissimilar and scler^tized structures. They are covered by a spicular sheath made of circular muscles and are supported by spicular muscles (Fig. 28).

Discussion

he male reproductive system of C. haukingi shous characters similar to that of Brugia pahangi as described by Schachcr (1 962) and Setaria cerui by ftnsari and Basir (1964). •e 23

Schacher (1962) and Ansari and Basir (1964) described testis followed by seminal vesicle but in this case testis is folloucd by v/as deferuns and the latter leads into wider seminal v/'isiclo which opens into cloaca through a muscular ejaculatory duct. Cobb (1905) and Rauther (1909) described hair like processes in the lumen of vas deferens but they are neither described by

Schacher (1962) and Ansari and Basir (1964) nor they are seen in C. haukingi.

FEHALE REPRODUCTIVE SYSTEM

The female reproductive system consists of paired ovaries, oviducts, receptaculum seminis, uteri and a common uterus, vagina and vulva.

Ovary

The jvarios aru didolphic and opisthodeIphic.

They are coiled structures. Each ovary consists of an outer epithelial layer and an inner germinal chord. A small cap cell is present covering the-: blind end of ovary (Fig. 29), Each ovary has two distinct zones the germinal zine and the growth zone.

The germinal zone starts just behind the apical cell. This area is the area of rapid division. It starts as a single cell but the number increases as it proceeds backwards. The cells arc spherical with a 124

large nucleus and a nucleolus inside (fig. 30 ). Thyse cells are enclosed by epithelial layer. This is follouedr-

by the grouth zone.

The grouth zone is relatively longer than

germinal zone. The cells filling up this part are kniun as oogonia. These oogonia are covered iver by a comparatively thicker layer of epithelial cells (Fig. 30).

Oviduct

Each ovary is fallowed by the oviduct. The wall of the oviduct consists of an outer layer of circular mus-

cles find an inner layer of columnar cells (Fig. 30)'

Uterus and receptaculum seminis

The oviduct of each side is follnued by uterus.

At the junction of oviduct and utorus the tube becomes a little enlarged f :)rmlng receptaculum seminis for storage of sperms. The walls of receptaculum seminis ore made up of irregular columnar cells with nuclei in the basal part. The columnar cells are lined externally by a membrane of epithelial cells (Fig, 3I ). The receptaculum seminis narrous diun and proceeds as uterus proper.

The two uterii run anteriorly and unite forming common uterus. The ualls of uterus arc made up of lou, rectangular epithelial cells. The lumen is full of microfilariae, in various stages of development (Fig. 32). 125

As it proceeds anterinrly its ualls become mars and more thick forming muscular uagina.

V/sgina

The vagina consists if tuio parts, the part close to the uterus is knoun as vagina utcrina and the part opening to the outside is kniun as vagina ve^ra or true vagina. The ualls of vagina uterina are made of columnar cells surrounded by circular muscles (Fig. 21).

The ualls of vagina ve^ra are thick with an inner layer of columnar cells and an out^r layer of circular and oblique muscles (Figs. 32 33).

Discussion

Different workers gave different opinion regarding the origin and existence of cap cell present at the blind end of ovary. Chituood (1 929) described this cell as a part of ovarial epithelium. I^usso (1930) gave a different view. He said that the cap cell is an undifferentiated germinal stem cell which proliferates both qerminal and epithelial cells. Ansari and Basir

(1964) found it underneath the epithelial cells covering the germinal zone of ovary. The author believes it to be a cell of distinct origin.

The receptaculum seminis is another point of controversy. Hagath (1916) believed it to be a part of oviduct while Chituood (1933) described it as a 126

modified part of utnrus. The author is also of the opinion that this part is a modification of uterus as in young specimens this part is distinctly set off from the ovaries and uteri but in mature females this constriction is lost and receptaculum seminis becomes confluent with uterus. This gives an evidence that seminal receptacle is a modified part of uterus.

Fundamentally the female reproductive system resembles both the parasitic filariae B. pahangi and

S. cervi so far histologically studied. Fig. 16. T.T. shouino layers of cuticle of body.

iq. 17. T.3. shouinq hioh chords of sub-cuticle,

musr^uI'.'^r '"art of oosociha, us nod sotnato-

o-isonhaqpol nusrles. 127

CU.

o mus oes. m som.oes.mus.

Fig. 15

20/U

Fig. 17 12t

Fig. 18

muse. mus.oes. lat.ch. o IT) som.oes.mus.

v.ch.

Fig. 19 F"ig, 20. T.S. shouing intestine and somato-

intestinal muscles.

Fig, 21. T.S, shouing glandular part of

oesophagus and u?gina uterina. 134

dorsch. cu. muse. o ui

Fig. 20

gl.oes. som.oes.mus

Fig. 21 Fig. 22. T.S, shouing nerve ring around muscular

part of oesophagus.

Fig. 23. T.S. shouing ronnective tissue Filling

up space body wall and

oesophagus. 13.0

O m

Fig. 22

to con.t.

Fig. 23 Fig. 24. T .S shouing germinal and growth zones

of tebtis.

Fig. 25. T.S. shouing uas deferens. 134

s

V. ch.

Fiq. 24

O vas.def. U)

Fig. 25 Fiq. 26. T.5. showing seminal vesicle,

Fig. 27. T.5. shouinq nliaca. 155

ion ino

Fiq. 26

muse. o If) dej. sp. sp.s. latch. rect. Fig. 28. T.3. shouing rectum, ductus ejaculatorius,

and spicules uith sheath.

Fig. 29. T.-i. shnuiinr cac cull of ovary. 157

o lO

Fig. 28

uo>

cap c

Fig. ?g Fig. 30. T.3. shouing /ones of ovary and oviduct,

f^ig. T.5. shouing rsceptaculum semim's. 134

Fig. 30

.ov. germ z.

ov. gr.7.

oogn.

Fig. 31 Fig. 32. T.S. shouinq uterus.

Fig. 33. T.S. shouinq ur^gina vera, 135

saoi6

Fig. 32

•saoiB

•jaA'6oA 3. Histochemistry

(i) Adult

(a) Acid phosphatase

(b) Alkaline phosphatase

(c) Adenosine triphosphatase

(d) Carboxyl esterase

(a) Acid phosphatase

Results:

The cuticle an.j the musculature shou feebly positive reaction Tor acid ohosphatase activity.

In the alimentary canal, the oesophagus, the intestine and rectum are strongly positive for acid phosphatase.

In female reornductiue system, the germinal and qrouth zones of oua rios shou a moderately positive reaction and the epithelial cells covering the nvfries shou a strongly positive reaction. The columnar cells of receptaculum seminis shiu a peculiar type of reaction,

The entire cell of the columnar epithelium does not exhibit the reaction but only some longitudinal strand like structures inside the cells shou a positive reaction for this enzyme. The wall of the uterus, vagina uterina and vanina ve/ra shou strong positive reaction. 137

In male, only the alimentary canal shows a positive reaction for acid ohosphatase activity(Figs.34-41)

Discussion

Acid phosphatase has been detected histochemically in sites associated with absorptive, secretory and excretory functions and so attention has been paid to the localization of nhosphatases in relation to these functions. For instance, in Hymenolepis diminuta

(Dike and Read, 1971a,b) and

(Nansour, 1959; Probert and Luin, 1974) the tegument uhich performs absorptive functions, shows a strongly positive acid phosphatase activity. On the other hand in Ascaris lumbricoides nutrients are absorbed from the intestine (Sanhueza, Palra, Gberhauser, Grrego,

Parson and Salinas, 1963), and this organ shous a strong phos'^hatase activity. In -^any other nematodes, the lurninal surface of intestine is rich in acid phosphatase indicating its involvRment in absorption of nutrients, as ue11 as secretory functions.

The hypodprmal acid phosphatase has been demonstrated histochemically in doo heart worm,

Dirofilaria immitis fYanaqisaua and Koyama, 1970).

Similarly in C. haukini^i, a positive reaction in alimentary canal proyeg that it is actively involved in absorptive and secretory functions. 138

The glandular surfaces of female reproductive system shou a positive reaction and thus it apnears that acid phosphntase is associated with secretory function. In ovaries a moderately positive reaction is present in the cells of qerminal as well as growth zones which are actively engaged in multiolication.

(b) Alkaline phosnhatase

The alkaline phosphatase was found to be absent in C. haukingi adult uorm. Fig. 34. Photomicr-jqraph of T.S. of adult '3houing

acid phosphatase activity in epithelial

cells, qerminal and qrouth zones of

ovary and intestine. X 400.

Fiq. 35. Photomicroqranh af T .' 3 . adult shauing

acid phosc.iT'^'j^ d ^ J activitv in mlunnar

Cells of rh;c(?r!taculun seminis, X 400. 139

£ n

•Z-ylB 'AO

'j'S-

•UAdS-J Tig. 35. Photomicnqraph of T.3. of adult showing

acid phosphatase activity in uterus. X 400,

Tig. 37. Photomicnqraph pf T.3. of adult shouing

acid phosphatase activity in vagina

uterina, X 400. 140

ut. Fiq. 38. Photomicroq]3Dh of section of adult

shouing acid phngphatase activity in

vagina vera (oblique SRction). X 400.

Fig. 39. PhotomicroL.raDh of T.S. of adult shouing

acid phosph^tese activity only in

intestine jf male. X 400. •^-le 141

•••fe.

--J.

r Ti int.^ 1

k Tigs. 40 41. Phot Tpi rrograohs Df T.3. of adult

shouin^^ arid phnsonatase activity

only in Intestine of male. X 400. 142 (c) Adenosine triphosphatase

Results

The cutiriB, sub-cuticle and musculature shou a moderately positive reaction for this enzyme. The oesophagus shous a feeble reaction uhile the intestine is strongly positive for this enzyme.

In the female reproductive organs, the qprminal as uell as growth zones of the ovary show a positive reaction. The glandular ualls of uterus and vagina shou a strongly positive rpaction.

The enzyiTG activity is found to be absent in male reproductive organ3(Figs. 42-45).

Discussion.

Adenosine triphosphatase occurs in areas uhere cells are involued uithor in absorption or secrrtion or transport of material or active tu1tiplication.

In C. haukin'^i the muscu l=iturs shows a positive reaction as this enzyme is synthesised in mitochondria of muscle tissue as demonstrated by Povyakel (1958) in pig Ascaris.

The intestine shows a positive reaction as it is involved in absorption and secretion as Ruitonberg

(1972) has found a strong activity in brush borders of 144

intestine of Anisakis sn. The nvarian cells shou a positiwe reaction ag the cells are actively inv/olued in division in germinal as iiell as qrouth zones of ovary in C. haukingi. Thn LIBIIS of uterus and vagina are positive for this enzyme as some secretory processes are going on in these structures. Fie. 46. Photomi cr 3qrapb of T.i. of adult snau^'ing

C3rboxyl esterase actit/ity in germinal

zone or ovary and intestine. X 400.

Fir. 47. Ph^tonicmor'' h ,f adult Jinuing

r^iboxyl c-,t' rc"3<= activity in nr DUth

zone Qp ovary. X /i-jT. Tig. 42. Photor"icn graph of L.S. of adult shouing

adenosine triphosohatase activity in

qrouth zone of ovary and glandular part

of oDSophanus. X 4GO.

ig. 43. Phot ornicro'-^r'p'^ of L.S. of adult shouing

adenosine tripmsohatase activity in

germinal z nne of lUr-ry aod rectum. X 4Q0. 145

i Fig. 44. Photomi cnaraph of L. 3 . nF adult shouing

adenosine trinhogphatase actii/ity in

walls of uaqina. X 4.00.

Tig. 45. Photominronrarjh of L.3. of adult shouing

adGmsine tri ohosphatage activity in

cuticle, musculature and v/uJva. TOO, 146

-vcjg.

"T'J (d) Carboxyl nstorase

Results:

The body uall shows a feebly positive reaction.

The lateral excretory channels shnu strongly positive re action.

Regarding the alimentary canal, only the intestine shows a positive reaction.

In the female reproductive system, the germinal zone and epithelial cells covering the ovaries, shou a positive reaction. The walls of oviduct, the luminal epithelium of uterus and inner glandular lining of the vagina show strongly oositive reaction.

In the male reproductive system, lining of epithelial cells, covering the testis, psoecially of germinal zone are highly positive for narboxylesterase activity. The epithelial cells of seminal vesicle also shou strongly positive re action/Figs. 46-49).

Discussion:

Workers like Boqitsh (1956) and Halton (1967) fouhd carboxylic esterase in tegumental regions and suggested that this enzyme takes part in nutrient transport through tegumental regions. They found the esterase to be nresent in oesoohagus and intestine and 148

presumed that this enzyme takes part in extracorporeal digestion. The presence oF this enzyme in teoumentary and digestive system has been demonstrated by Bogitsh

(1 966) •

In C. haukinpi, the intestine shouing positive reaction goes to prov/e that it takes some part in digestion. A positive reaction shoun by epithelial lining of male and female reproductive system affirms the view that these linings take part in nutrient transport.

A strongly positive reaction exhibited by lateral excretory channels Further support the vieu that this enzyme takes part in the transport of the excretory fluid. . - 149

..M ov.germ.z.

\ • Fiq. 48. PhotnmicrDQraph of T.5. of adult shouiing

carboxyl esterase activity in uterii. X 400,

^iq. 49. Photornicn "^rcrj'i )f T.3. nf adult shouinq

carboxyl 'ist-iT-iar-- act lu ity >.n epithelial

cells of tpstis. X 400. 150 (ii) Microfilaria

(a) Acid phosphatase

(b) Alkaline phosph.'tase

(c) Adinosine triphosphatase

(d) Carboxyl esterase

(e) Aryl sulphatase

(a) Acid phosphatase

Results:

The distribution and intensity of acid phosphatase

activity in the microfiJ aria of £. haukingi is presented

in Table 6. Smears incubated in substrate free medium

showed no reaction product in microfilaria.

Strong acid phosphatase activ/ity uas seen in

''lungebi Ide, excretory vesicle, G^ cell and anal vesicle.

Comparatively feeble activity uas observed in the first

feu cells of the nuclear coluTin, sub-cuticular cells

of anterior half of the body, cells and last

tuo cells of ^hp nuclear colunn, oresent near the tip

"of the tail(Fiqs. & 51 ) ,

Discussion •

The distribution of the acid phosphatase activity

in microfilaria is so snecific that it offers a parameter

for dis t inqui s h i n o ths pp^nera, s 'ecics and even the strains of filarial unrms. Thus several investigators distinguished the species of filarial uorms by localizing the distinct patterns of acid phosohatase activity in circulating rnicrofi lariae. Chalifoux and Hunt (1971),

Balbo and Abate (1972) distinguished the species of

Dirofilaria from Dipetalonema even in mixed infections.

Redington, Plontgomery, Gervis and Hockmeyer (1 975) distinguished histochemically the microfilariae of

Brugia malayi from B. pahangi. Teruedou anri Huff (1976)

localized this enzyme in LJuchereria bancrofti. Hmar

(1977) confirmed the observations of previous workers and localized the enzyme activity in developing stages in the vector of all the four filarial worms viz.,

Uuchereria bancrofti, Brugia malayi, 8. pahangi and

Dirofilaria immitis.

Braun-f^unz inqer and Sputhgate (1 977) uent still further by di f fe r f? nti at ing the four different strains of Onchocerca volvulus by localizing aciri phosphatase in the micro filari ae (TableT)•

The different areas positive for acid phosphatase indicate that this enzyme activity is present only at metabolically active regions. It is supposed that acid phosphatase activity is associated uith certain important functional processes such as absorptive, phagocytic, excretory or secratory activities of the living cells. 153

The microfilaria of C. haukinqi shous strong activity at the anterior end correspondinq to PlungebiJrie or amphiis, probably due to its absorptive function.

Similarly the strong reaction in the excretory and anal vesicles indicates the involvement of the enzyme in excretory or secretory processes. The significance of enzyme activity in the C^, R^ and R^ cells, and the first feu and last tuo cells of the nuclear column is not clear. It appears, however, that the enzyme is involved in cell proliferation, synthesis, secretory and absorptive functions.

The intensity of activity of this enzyme at

Plungebilde is very strong and almost similar to that of

Dirofilaria immitis. Similarly the activity in the excretory vesicle resembles that of B. malayi,

U. bancrofti and immitis but not B. pahangi in uhich comparatively lou activity has been recorded. Here

Innenkorper showed a strong reaction corresponding to that of U. bancrofti.

Anal vesicle shous strong activity for this enzyme.

This structure shous equally strong activity in all the described species. Uariable intensity of enzyme activity uas shoun by Schuanzgehilde and sub-cuticular CGlis in the different specics. 154

4-> P CO •H rH -P D O u Q) CO •p Oi -p + (-! + (-< + (-. CD OJ n + + o a I + O I Q jr CJ cn -p 1 Xi X D CU Q) -p cn CJ C — » — 3 1 c N Q) o "O C T3 CD (D r-H + f-i + p + (-( U J -P 1 + o o I + o I T3 •H r-- - — — — T) -y CTi 0) 3 r— P C CD CD O JZ a •rH U p -P « a :0 + -P c U E O Q) c + CD O u C CD c >- -P 1—1 -P ti- - _ - - •H CQ —' p O CD -P cn -P + + o m CD U ¥ + + (H -t- M + M CO •H p -H -t- + O O 4- O I O O o u m CD Q} X -p CO a ixj ro CO _ _ ^ _ -p CD in XI ^ CT. + u u x: Q) P C 1 I (-1 + o + o u. X) Uj -H o (0 •H 2 • P o P ^ ^ _ , x: o CD Q cn XJ cu • 1 "D Tl •P •H Xi + CJ C m + (-1 I •P a. o c • D UD C

CD r-H >> XI (U cu CT rH 1- cn ro" o m •P| £ c •p u 3 CO £ •Tj Q) Cd j •rJ r> c CJ1 a. cn D • (-J 11 c-'l iX) cJl 155

cu 3 u 4-> + M f-4 + u u LJ CO + o + o I -f G + O t •—1 D CD 'n U

1 N CD C T CD' —( + p-1 p 3 •H O I + o + o -C X) o CD cn U

CD .-H O r-|• H + CO CD + + (H + c CD + + o + CC 25 oH m -o _ O- X) CD J cn i-i Q) C G r- to + -iC + f-( + ro ro 03 c + O + QJ 4-> B Cl- OJ cl m u ^ c a c 0 3 c TO D 1- X I—1 >. o (D -p i + m O + (-1 + (h (-1 + t^ + n f-l •H + o + O + O I + o + o + u cn X •H l-J (D cr + (-( u p c + (-4 (-1 + o + o CD•r H + 0+0 z IH

Q •a 1—( CD •H •H CD n fH + f-l + -P m n I f o + •H •H + cn P C o •H 2 CL cn O ^ - >. CD CL CD rH G>• H •p CO OD •H C -P r-l -p CD •H • ri U n •rH V CO •H f-i 4-1 t-i cn CO a> U CD Cl— r-H col o CD CD © U n •H •r-l Q. D- (D f-l Cl- -P rn XT o o •r-l + O c F + D CO •r-l b + + H 1 3 XJ a cn X CI r) D •P c r-t Ol OJ CM -p 3 c cn ra CD (-1 tJ CD c JSC rH o o X CD C •r-t + .H + -P rH •r-l . O U C Ol •D Q) CD CJi CD c CO O -4-' -P c >-H CD -H o m cn CO o O CO C n • O O rH X 3 Q) --—- O- a CO 3 Ci_ CO U Ci- -P O t> •H CD JZ CT> X) u -H Cu. c rH + •P O n ^ CD + f-i + X CD O + o + cn c Cl- •H o o (0 L3 r-l -H O' •p CO X ra c -p CD N •iH D TD X3 CD -H CD o rH 0) > M U cn •H C CO -P XJ O • O O CO "D CD •H -P O c O -P rH -p CO N C CJi c C Q] c -P 03 (-1 CO E •H •rH C (-1 cu 3 C to CD cr X •rH > o C CD C|_ + o + c CO -P (0 d 5 •rH CD -H •H f-l X] u > aa 1 M Cl_ CO •rH C CD CO O M -P u m Q 3 •rH u CD C (-1 o CD Ci- C^ -p C :C X CJ o l-H ^ to C P ffl to C' o (0 -—- >. cn -H CO M Xj X E -p CD C CJ o (0 CO CD -P X CD X •rH 0) u + 0 t a U (H -H + CD + f-l p -P O CO CO u to + X + o + O 1 (D X o t-H X --1 -p •-H 3 x: •H UJ > D. D C;- H- e (u dj O CD Q n X "D U P CJI U 4J •H CD •r O M C CM -P u •H a) -r-l r ^ •rH cE ^ (-1 U c (J1 -P B p O •H • CD f-l Ct_ CJI a cn •a o c o •H CO -rH >-03 D- CD CD •H (-1 (D C rH > 5 r-C X ai •rH -H XI 0) + V p CO C 'H -P CO 1 + U —I -P r -H ^ ra c + o + a 01 p tn 0, Qi 4-) O CD 0) 2 rn U. 3 ^ + tt) o + + [XI LJ o + + I Fig. 50, Photomicro qraph of lonn form o^ micnfilaria

shouino acid phosphatase activity. X 900.

Fig. 51. Phot iniur-ipr' ph of short form of

micri'-MlcMiP nhnuing acid phosnhatase

activity. X 400. 157

t 158

(b) Alkaline phosphatase

Alkaline phosphatase uas found to be absent in microfilaria of C. haukingi. 159

(c) Adenosine triphosphatase

Results"

adenosine triph isphatase is intensely present in the cephalic end, cxcretory vesicle and G^ cell.

The sheath also shows a feebly positive reaction(Fig. 52)

D iscussion:

Adenosine triphosphatase is associated uith energy metabolism, pembrane transport, muscle tissue

formation and absorptive functions. The strong ATPase activity, exhibited by the cephalic end, excretory vesicle and G^ cell indicates the functional activity and energy rretabolisr" in thesc^ structures.

The cophaiic end takes part in absorption or membrane transport of food material; the excretory vcsiclo shous a strnnq reaction for this enzymo, as it is associatpd uif- tbt metabolic processes. G^ cell shous activity, as it is rich in chnmatin materia] and is first to divide during development. '006 X 'A^T^^T^oe aseq-eqcl

-soq-dtjq. butsguepe c>UTnoi,s biatjixjojqtuj

JO ujjoJ 5uo-[ JO L|deJboaoTUJo::^oq(j • 'OTj 160

'.v. 1 161

(d) Carboxyl ostcrase (non-specific)

Re suIts

An intense nnn-soecific cirboxyl esterase activity uas 'observed in the cenhalic region of microfilariae from anterior tip to the nerve ring, in the excretory vesicle, anal vesicle and one or tuo cells near the tip of tail. A feu sub-cuticular ce Us also shoued the carboxyl esterase activity(Fiqs. 53 & 54).

Discussion:

Carboxyllc estrar hydrolases (carboxyl esterases) are a large group of non-specific hydrolases acting at pH optima betuecn 5.0-9,N on simple short-chained fatty acid esters. They r,rn both hydrolytic and synthetic in action. Houiever, ib is true, as layers (1960) has pointed out, that the "metabolic functions of the esterases are not completely knoun.

Tho strong esterase activity in the anterior part of the microfilaria indicates a possible function of tho enzyme in absorption and resynthesis of fatty acid esters. The enzyme activity in the nerve ring region might represent a cholinosterase activity since

oC-naphthy lacetate is also hydrolysed by cholinesterases.

Houever, the possibility that the esterases are involved in the synthesis of lipids, characteristic of nervous system cannot bo overlooked. 162

Similarly the i^cll knoun association of esterase

activity uith cxcrotory function in many animals, offers an explanation for tho intense enzyme activity seen in the excretory and anal vesicles of microfilaria.

In the cells of the posterior end of the microfilaria, esterases might be involved in cell proliferation or synthetic functions. Fig. 53. Photomicroaraoh of long form of

microfilaria ahouinq narboxyl esterase

activity. X 900.

Fig. 54. Photomicroqrar'h of short form of

microfilaria shouinq carhoxyl esterase

activ/ity. X QOO. 163 164

(s) Aryl 'Sulphatase

Results:

A diffusnd reaction all over the body has been obseruedJ^Figs. 55 & 56).

Discussion;

The entire body of the microfilaria, rather the nuclear column, showed reaction for nryl sulphatase uhereas tho Innenkorpor region gave a negative reaction in long forms and in short forris a diffused reaction is oresent along the entire length of the body including

Innenkorper . Fig. 55. Photomicrograph of long form of

microfilaria showing aryl sulohataao

activity. X 900.

Fig. 56. Photomicroqrrnh of short form of

microfilaria shouiino nryl sulphatase

activity. X TOO. 165

-A- 166

4. In vitro cultivation

All the ijn vitro experiments uere carried out at 22 C and at the pH 7.5 adjusted with the help of

0.4^ NaHCOj or Tris buffer and tested with universal i ndicator.

Experiment No." 1

Tyrode's solution (Tyrode, 1910)

Ringer-Locke (Locke, 1901)

Hank's BSS (Hanks and Wallace, 1949)

and Earle's saline (Earle, 1943) uere taken and supplemented with 20 or 50^ of inactivated bovine

SB r urn.

The microfilariae survived for 6-9 days, both in

Tyrode and Rinqpr-Locke uhen supplemented uith 20/S bovine serum. The survival time became 11-13 days uith 50/S serum supplement.

The survival time did not improve much in Hank's

BSS and Earle's saline. Uith 20w serum supplement the survival time uas 5-10 days and uith 50^' the survival time became 10-12 days.

No grouth or development could be observed.

Usually the microfilariae remained very active for

4-5 days and then they started becoming sluggish and dying gradually. 167

Experiment No. 2

Chemically defined medio, NCTC 109 (Evans, Bryant,

Fioramonti, f^cQuilkin, Sanfor-, UestFall and Earle, 1956;

McQuilkin, Evans and Earle, 1957).

Eaqle's MEM (Eagle, 1959)

CnRL - 1066 (Parker, Castor, NcCullock, 1957)

Grace's Gi^A (Grace, I95f0 uere used supplsmentinfj

^ uith 20% or 50% bovine serum,

(Microfilariae survived for 10-12 days in NCTC 109 and in medium 199 uith 20% serum supplement slight increase in length (6 jj) occurred.

The survival time uas reduced to 5-5 days uhen serum supplement u's increased to 50%, It uas noted that there uas a gradual decror'se in the pH of the medium uhich became acidic (t)-4) after 5 nays. It is, therefore, concluder that either the microfilariae could not stand the acidic pH or the concentration of the media, uhich may be too rich.

The microfilariae survived for 14 days in ClRL-IOeS,

They remained very active in Grace's medium uhen supplemented uith 20% bovine serum and survived for

20-24 days. No growth or development uas observed,

50% serum supplement uas not used. 168

Experiment No. 3

Earle's saline, Ringer-Locke and Hank's B53

supplemented with lactalbumin 5%, yeast extract 2%.

In addition, 20/ serum of uhite rat or guinea pig or bovine or foul were used.

In Ear la's medium, having the above mentioner; percentage of lactalbumin and yeast, uhen supplemented uith 20^ uhite rat serum, the microfilariae survived for 12-16 days; uith 20'^ guinea pig serum the survival time became 15-17 days; uith 20^ bovine scrum, the microfilariae survived for 8-10 days and uith 2 0>o foul s'erum for lR-20 days.

In Rinnor-Locke and Hank's BSS the survival time did not improve. The range of survival time is almost the s'me.

Experiment No. 4

This cxneriment u-^s done by using Eagle's nE*^, nedium 199, NCTC 109, C'^^IRL-IOee -"^nd Grace's Gi'lA. The microfilariae kept comparatively more active, the survival time uas increased and sometimes there uas little increase in size.

liihen Eagle's NEn, having lactalbumin (5%) and yenst extract (25^), supplemented uith 2 0^ uhite rat serum, uas used for cultivating microfilariae, they survived for 7-10 days but no grouth or development uas 169

observed; when guinea pig sarum was supplemented, the microfilariae survived for 10-11 days, they kept quite active uith 20^ bovine serum and survived only for 6-8 dcys. Uith foul serum supplement the survival time increased to a maximum period of 20 days.

Thereafter the microfilariae started becoming sluggish and dying gradually.

The results in medium 199 and NCTC 109 were almost similar. IMCTC 109 uith 2% yeast extract and 5% lactalbumin, uhen supplemented with 20% uhite rat serum, the microfilariae survived for 12-15 days and in Medium 199 thoy survived for 12-16 days. In both, slight increase in length (4 yu) uas observed; uhen

NCTC 109 and medium 199 were supplemented with 20';^ guinea pig serum, the survival time became 17-19 days in both the media. nicrofilariae kept very active and perhaps due to the urigqling of microfilariae, the sheath at the anterior end got broken. An increase of

6 yu in length was observed; uhen supplemented uith 20% bovine serum the microfilariae survived for 10-12 days in NCTC 109 and 8-14 days in medium 199; uhen supplemented with 20% foul serum, the microfilariae survived for 15-20 days in NCTC 109 and for 17-20 days in medium 199.

Yeast extract (2%) and lactalbumin (5%) uere added to CnRL-1066 and Grace's medium also and then 20/^ of different sera supplements uere given for cultivation of microfilariae. In CflRL-1066 uith 20% uhite rat seru^r), the microfilariae suruiued Tor 14 days; uith 20% guinea pig serum the microfilariae suruiued for 18-22 days, breaking of sheath and increase in length (6 /u) uas J observed; with 20% bovine serum they survived for

12-13 days; with 2 0% foul serum the microfilariae survived for 20-24 days.

The Grgce's GMA, gave the best results. When supplemented uith 20% white rat serum, the microfilariae kept very active, the shenth usually got broken. They survived for 14-18 days; with 20% guinea pig serum supplement, the microfilariae survived for 20-22 days; with 20% bovii.e serum supplement, the- microfilariae survived for 12-15 days and uith 20% foul serum suoplement the microfilariae survived for 20-28 days.

Experiment No. 5

In this experiment the microfilarioe uere cultivpted in Grace's G(^A and CnRL-1066 supplemented uith 20% crou serum, crou blood ce lis (separated by adding citrate, diluting and centrifuging), minute fragments of Aedes larval gut, 0.5% Aedes (pupal) haemolymph, 1.0% glucose and 0.5% CEE.

The survival time increased to 25-30 days in

CnRL-1066 and 43-45 days in Grace's GI^A. In Grace's

GNA, the microfilariae survived for the longest time. 171

most of them showed broken sheath, vet there uas no true ecriysis, a feu became shortened and thickened with broken sheath still intact. i^o true deunlopmental stages could be obseruod.

Experiment No. 6

nonolayGrs if white rat embryo, chick embryo, heart, kidney and liver (uhite rat prenatal) uere prepared and overlaid uith Hank's P3S, lactalbumin 5%, yeast extract 2/ and uhite rat serum IT"^.. The microfilariae survived for 5-7 days and then '^ost of them nenetrated under the monolayer and died. c3ome of them uero found lying dead under the monolayer even after 24.0 hours. discussion:

The microfilariae of Chandlerella haukingi survived for about 10-12 days in NCTC 109 and medium 199 uith 20% scrum supplf^ment, uhile the microfilariae of

Mrofilaria immitis (Earl, 1959), survived for 4 days in medium 199 alone, for 43 days uith 10'/. scrum supplement and for 61 days uith serum supnlomont.

In the present case, survival time of C. haukingi decreased to S-6 Hays only uhen the serum supplement uas increased to 50%.

The survival time of C. haukinqi microfilariae is comparatively longer than that of D. immitis microfilariae ''jauiytr and U'^instpin, 1961) uhen cultiv/ated

in V/itro in Rinqer and Locke's solution uith different

supplemonts, Sauyjr and Ueinstein (1951) had used

Krebs-Kinger phosphate i/ith glucoso and ^ .Sf- sodium

caseinate and C. haukingi microfilariae uere cultured

in Tyrodo and Hinq^r Licke's solution with 20% bovine

se rum.

The microfilariae of D, immitis surv/iued for

7 days (Sauyer and Ueinstein, 1161) in NCTC 109 and the microfilariae of C. haukingi survived for 10-12 days

in this medium.

The survival time of microfilariae of C. haukingi

in GPIA with 20) sprum supplement remained only for

20-24 days but the microfilariae of naracanema formosana

(Uood and Suitor, 1966) dev(^ loped to infective stage

in GnA supplementpd uith D.S^ insect haemolymph and 1

foetal bovine serum.

Bcjsides all these, a number of other rombinai-ions , chcmically defint d mfdia, ^upnlemented uith various nutrients and TC monolayers, overlaid uith growth media, having Hank's 653, lactalbunin, yr.ast extract and serum, have been used for cultivatinn vitrn, the microfilariae nf C. haukinni. Thj results uerp obtained uhen cultivat'-d in G!'1A supolemented 173

uith 20f>' rrou Sf^rurr, nrou bloTd Cfjlls, minute fragments of Aode3 larval qut and 0.5/^ Aedes ^pupal) haumolymph,

1.0% glucose and 0.5'/^ CEE. In such a medium the suruiual time increased to days. 174

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ABPREUIATI3NS

a. p. anal pore b. rn. basal membrane c. s. cephalic space cap.c. cap cell ci r.mus. rircular muscles con.t. connectivye tissue con.t.sup. connective tissue support cu. cutic le cu, s. cuticular striations cu. th. cuticular thickenings d . e j . ductus ejaculatorius dors.ch. florsal chord e. p. excretory pore f. c. n. CO 1. first cell of nuclear column fib.1. fibrillar layer fib.lam. fibrillar lamella fib.z. fibrillar zone fibr.1. fibrous layer

G^ cell S gl.oes. qlandular oesophagus h. hook int. intestine i nt.lu. intestinal lumen

1.c.n.col. last cell of nuclear column E24

1. gn. lateral ganglion

mat. 1. matrix layer

m f. microfilaria

mu3.oes. muscular oesophagus

muse, musculature

n. r. nerve ring

nu. nucle us

0 . oral ring

obi. mus. obligue muscles

oogn. oogonia o\j. ovary

a\j. d. oviduct

o\y. germ, z . ouary germinal zone o\/. gr. 2 . ouarv qrouth zone p. t. pharyngeal thread r.sem. receptaculum seminis

re ct. re ctum re ct.c. rectal cell s . spi ne sem.ves. seminal vesicle som.int.mus, somato intestinal muscles som.muse, somatic musculature som.oes.mus . somato oesoohageal muscles sp. s. spicular sheath spl. z. splanchnic zone 225

spmt gon. snRrmatoQonia spr. sf.ierm sprz. snermatozoa t. testis t. germ, z . testis germ zone t.gr.z. testis growth zone ut. uterus

V ag. u aoina wag. ut. vagina uter ina uag. v/er. V/ agina uera vas.def. \jas deferens SUI^NARY

The thesis embodies the results of the studies on Chandlerella hawkingi, a filarial parasite of Indian jungle crouj, Corvus macrorhynchos (Uagler). Only four aspects, morphology, histology, histochemistry aad in Vitro culture have been taken up.

The morphological studies include a re description of adult uorm uith an addition of a feu minor details in the adult. The structures described in microfilaria are the nuclear structures, cephalic structures, pharyngeal thread, Innenkorper and also occurrence of tuo forms of microfilariae in blood of crou.

The nuclear structures hav/e been studied uith special reference to nuclear landmarks which are of great taxonomic valuo.

Sometimes the nuclear structures do not suffice for identification of genera and soecies, the cephalic structures, pharyngeal thread and Innenkorper have also been studied.

It has also been proved that the tuo forms of microfilariae, the long and the short, present in the blood of crou belonc to the same soecies, C. haukingi. 265

The stereosnan studies of the cuticular structures

of the adult and the microfilaria haue been done as these

are of great taxonomic importance in classification of

uorms.

The histological studies include the study of

histological features of the body uall, musculature,

alimentary canal and reproductive organs as some of these structur- provide a taxonomic tool at various levels,

iiq histochemical -^^.tudies, four enzymes, viz;

acid phosphatase, alkaline phosphatase, adenosine triphosphatase and carboxylesterase have been localized

as these enzymes are attributed to different functions and their distribution is very specific. Thus the

localization of these enzymes hr-slos in determining the various functions assigned to different organs and parts of the body of adult uorm.

In microfilaria, five enzyn-93, acid phosphatase, alkaline phosphatase, adenosine triphosohatase,

carboxylesterase and aryl sulphatase, have been studied.

The distribution of these enzymes is so specific that their patterns help in identification of different genera and species. 228

In the prnsent u.'ork only nicnFilariae of

C. haukinqi have been cultiv/ated in vitro, in vieu to study thoir dcvelopment outside thn body of tho host.

These studies are important even for tfjsting the drugs on parasite alone and also for collection of E5 products uhich may serve as functional antigens against filariasis.