PART. III.

STUDIES ON IN SOME SNAICES 140

INTRODUCTION

Hepatozoon is an intra-corpuscular non-pigmented parasite of vertebrates. Levine,et al»,(1980) placed Danilewsky, 1885, Hfepatozoon Miller, 1908 and Labb^,1894 in three distinct: families viz,,

Haemogregarinidae Leger,1911,„ Hepatozoidae WenYon,1926 and Wenyon,1926 of the suborder Adeleina under the Class Sporozoea, The characteristic features of the genus Haemogregarina Danilewsky,1885 has • been given in Part II, page nimber. 151, Genus Hepatozoon Miller,1908 is characterised by the presence of schizogony cycle in cells- of the internal organs ( liver, spleen, kidney, lung and bone- marrow etc. ) of vertebrate hosts. At an interval of several generations, some merozoites enter: erythrocytes or: leucocytes as in the case may be, and develop into gametocytes. Sporogony stages are knovm to occur in hosts. Zygote develops into oocyst stage. These increase in size and become ^quite large. Oocysts contain num.erous sporoblasts and ultjjnately to sporozoites.

Genus Karyolysus Labbe'', 1894 is distinguished from Haemogregarina and Hepatozoon' on the basis of the following characters. 141

Schizogony occurs in the endothelial cells of the blood vessels and the gametocytes enter the red blood corpuscles of vertebrate hosts., Sporogony occurs in invertebrate host (mite) v/here large oocyst deveops containing sporoblasts. These escape from oocyst as motile vermicules and enter the egg in v/hich these become sporocysts within v/hich sporozoites are-: developed.

The above three genera have been reported to occur in the peripheral blood of cold-blooded vertebrates, Gametocytes-of these genera do not always provide a reliable clue to their generic differentiation. As a result, most of the snake haemogregarines have been placed under genus Haemogreqarina although in many cases erythrocytic schizogony - and sporogony of these parasites are unknown.

Many species of Hepatozoon have been described from v/arm-blooded vertebrates but there are very- few reports about their pccurrence in cold-blooded animals.

In India/ there has-been little work on Hepatozoon. A syrvey of Hepatozoon in ophidians from some parts of West Bengal was undertaken. Results of this survey are given in this chapter.

The geographical and hostal differences relating to the haemogregarines described in this work appear to be enough to distinguish them from previously named species given the present ver^^ imperfect state of our knowledge of this group. 1'42

REVIEVI OE LITERATURE

Hepatozoon was first seen by

Bentley (1905) in leucocytes of dog from rndia,and it was named Leucooytozoon canis by James(1905). Balfour

Sudan and named it.Leucooytozoon muris in 1906, Miller

(1908) founded genus Hepatozoon for a parasite of leuco­ cytes of rat and named it Hepatozoon perniciosijm,

PTepatozoon in Ophidia.

An ophidian Hepatozoon was discovered by Garnham (1950) from an African sv/amp snake,

Crotaphopeltis deqeni' which he named Hepatozoon minchini.

Hull Sc Camin (1960) described some haem.ogregarines in snalces and stated that these parasites should be placed under genus

Hepatozoon,all belonging to a single species, Marguardt

(1966) described Hepatozoon sp. in some snaEes in Southern

Illinois. Mkrtchyan (1967) described Hepatozoon" ophidia from the blood of Russian snake,Elaphe hohenackeri. Ball, e^t al., (1967) transferred Haemogreqarina rarefaciens to genus Hepatozoon on the basis of thee study of its life cycle,the material being collected from Drymarchon corasis' in Coliraa and Te^ic,Mexico, Clark & Bradford(1969) desc­ ribed five types of a haemoqregaine of the genus Hepatozoon in a single blood smear of a snake,Pituophis melanoleucus mj

catenifer collected from Pacific North West. Ball^et al.^ (1969) described Ifepafeozoon fusifex in Boa constrictor collected in Colima ,Mexico. Pessoa S:; Cavalheiro (1969a/ b) and Pessoa.et. CLI ., (1970) described the sporogonic stages of three species of Hepatozoon in water snakes of Brazil. Booden/et al.,(1970) described Hepatozoon sp. in Boa constrictor and its transmission to a lizard by mosquito vector. Oda.et^; al., (1971) observed Hepatozoon in a snake and its transmission. Pessoa Sc 3iasi(1973) described several species of Hepatozoon parasites of Br^silian snakes. Ball S Chao(1973) observed the sporogo- nous stages of Hepatozoon rarefaciens cultured in a Culex pipiens

WORIC DCN'fi IN INDIA.

Sinha (1980^ described Hepatozoon sp.

in a snake/itophiesma stolata collected from West Bengal.

Hfe (1981^ also recorded Hepatozoon'sp, Ih a rat snake,

Ptyas mucosus collected from Bongaon,24-Parganas,West

Bengal. 144

RSrERIALS

The following hosts were examined

for Hepatozoon Infection in their peripheral blood.

-'-• Pt.yQ.s mucosus (Linnaeus) This is commonly known as Dhaman* in Bengal. It belongs to Eanily Colubridae under Order Squamata. It inhabits plains,often in the vicinity of human habitations. It is diurnal,non-poisonous,timid and is capable of climbing trees. Itr is olive-green,brown,yellowish in colour- above with', irregular cross-bars black in colour on posterior half of boSy. Ventral side is yellowish in colour . Posterior ventrals and sub-caudals are edged black. Head is disiilnct from neck. Eyes are large with a round pupil. It is known as rat- eater althbugh it has no particular choice of food. rt is distributed throughout India, Indo-China ,Sri'Lanka and adjoining countries.

Five specimens were collected from Bongaon,24-Parganas,West Bengal and two of these were found to harbour Hepatozoon in their peripheral blood.

2» Anphiesma stolata (Linnaeus)

This snake is known as striped keel-back. It belongs to Family Colubridae an under Order Squamata. Body is olive-green or brown in colour above t45

with black spots or reticulated cross-bars intersected by- two dorso-lateral yellow stripes. These stripes were well marked on the^hinder part of body. Ventral parts are white sometimes with a black spots on the side of each ventral shield. Head is olive-green in colour and the shields are edged with black. It . : is diurnal^non- poisonous and can be handled easily. It feeds chiefly on frogs and toads. I-tj:. is distributed throughout India, Sri Lanka and adjoining countries.

Six speciemens were collected from Chakdah, Nadia/West Bengal and examined. Two of these were positive for Hepatozoon in their peripheral blood,

3. Naja naja (Linnaeus)

This poisonous snake is popularly known as'Gdkhura'in Bengal. It belongs to Family Slapidae under Order Sguamata. It is olivaceous or brovTnish to black in colour. Head is not very dis­ tinct from neck. Neck region is dilatable which forms hood. Nostrils are large. Scales are placed obliquely, Eyes are round. It; feeds on rats,mice,toads and frogs. It occurs in Bengal,Eastern Himalaya and in Nepal. 146"

Pour specimens were collected from

Baruipur,24-Parganas,West Bengal, India,for examination and

one was foxind to harbour Hfepatozoon in its peripheral blood,

METHODS

The following methods were adopted for the prepararation of blopd smears and tissue sections.

Blood of snakes were obtained either by clipping the tail region or from the facial vein.

Infected snakes were killed and small pieces of tissue from liver and lung were fixed in Bouin's•and

Camoy's fixatives.

Blood smears were stained in Eeishman's and Giemsa's stains.

Tissue sections were stained in Heidenhain

"^ iron-haematoxylin and haematoxylin-eosin stains.

Measurements of parasites were made from

fixed and stained materials and Camera lucida drawings were made,

Photomicrographs were also taken in some cases. OB; •^S-iLAT I On 147/

Hepatozoon in Ophidia.

parasite No. 1.

Hepatozoon n. sp. (a)

(FIGS. 1 - 2fe )

OcKurrenoe

The parasite was encountered in the peripheral blood smears of rat snakes ( Ptyas mucosus Linnaeua ) collected from Bongaon,24-Parganas/West Bengal,Tndia.

Morphology

Parasite in peripheral blood were intra-erythro- cytic gametoocytes which were at various stages of develop­ ment.

Gametocytes (Pigs.l - 10.)

These were abundant in the blood smears.

Two distinct morphological types were found depending on their size,shape and staining properties. Sexes of these gametocytes could not however,be conclusively distinguiished.

Type. I. ( Figs. 1-4.)

Young individuals (Pigs.l - 2 ) were oval or bean-shaped measuring 5.5/um - 9.2/Jm X 1.67um - 3.8/um

(average 7 Aim X 3.2/um ). Cytoplasm was homogeneous, non­ granular and stained very faintly . Nucleus was central, non-compact and irregular in shape. It stained deep pink in colour. Average measurement of the nucleus was 3.2/um

X 2. 3 Aim • 148

Type. II.. As the development advanced, parasites were enclosed in a polar cap at each end which stained deep blue and fitted over parasite as a cup. Polar caps were very prominent xirith a maximiam length of 2.6 /am. Mature gametocyt^s ( Pigs. 3-4) measured 11/um - 13.2/um X 2.7 AMI - 3.3 Aim ( average 12/um X 3/am ), Cytoplasm was homogeneous, basophilic: and non-granjalar . Nucleus was compact, sub- spherical or oval,central or sub-central v;ith deeply stained chromatins. Average measurement of the nucleus was 3.3 /am X 2.6 /um.

Type. rr..

Young individuals (Figs. 5 -6j., were thin and narrow,one end lying broader than other. These measured 9.7 Aim - 11.2 Aim X 1.4/um - 1.6/um ( average 10.5/um X 1.5/um X. Cytoplftsm was homogeneous,non-granu­ lar and faintly stained. Nucleus was elongate or oval sub-central and occupied entire v/idth of cell body. As the development advanced, a capsule developed. Inside the capsule,parasite gradually assumed the shape of a charact­ eristic vermicular form. One end of parasite was sharply bent forming a hook like structure. Other end was broad or tapered slightly. Mature forms ( Pigs. 7 - 10) 4

5 nrr\

T 8 10 11 12

FIGS.I - 10, Camera lucida drawings of Hfepatozoon *H.Sf. (ou). FIGS.l - 4. Intra-erythrocytic gametocytes,T^pe I, FIGS.5 - 10. Intra-erythrocytic gametocytes. Type IX. PIGS.11 - 12. Normal erythrocytes* 149

measured 12/um - 16 Am X 2 Aim - 2.5 Aim ( average 14.2 Aim X 2.2 >um ). Cytoplasm showed similar features as in young forms. Nucleus was sub-terminal and deep pink in colour. Average measurement of the nucleus was 4.3 Aim X 2.4 AJtia,

Tissue phase. NO divisional stages were ever encountered in peripheral blood smears. Schizogony occurred in liver and lung of snake and these could be studied in sections:*

Trophozoites (Pigs. 13 - 14) Early forms as noted in the sections of liver were ovoid to sub-spherical measuring 4.4 Aim - 7.7 Aim X 4.8 Aim - 6.5 Aim ( average 6 .8 Aim X 5 .5 Aim ) . Cytoplasm was homogeneous,non-granular,non-vacuolated and light pink in colour with eosin stain. Nucleus was large, oval and deep blue with haematoxylin stain. Average measurements of nucleus was 3.3 Aim X 1.6 Aim. In sections,trophozoites were found surrounded by a clear empty space. Witl further development the parasite became sub-spherical tto oval and the nucleus divided into two daughter nuclei ( Fig. 15), 150

schizontsi ( Pigs.16 - 26)

Schizonts with two nuclei (Fig,15 ) measured 6.8 Am X 5.6/um. Cytoplasm was homogeneous,non-granular and took up eosin stain. Huclei were deep blue in haemat- oxylin. Average measurement of the nucleus was 2.4 Aim X 1.6yum. As the development proceeded,two types of schizo­ nts formed. Mature macroschizonts ( Pigs. 21 - 25 ) measured 10 /um - 13/um X 8/um - 11/um (' average 11.5 Aim X 9 /um) and produced two (Fig.27 );,four (Figs.21 - 24, 28 - 29 ) and eight (Figs. 25, 29) macromerozoites. Microschizonts (Figs. 16 - 20 ) were sxib-spherical to oval measuring 7,7/um - 11 /jm X 6 .5 /um - 8.8 /tim. ( average 9,5 /um X 7 .2 Aim ) with thirteen (Fig. 18);,sixteen ( Pig. 17), twenty four (Fig. 19 ) and thirty tv7o nuclei (Pig. 20). Ci^toplasm contained a reticular net-work of fibres with dark blue nuclei scattered throughout the cytoplasm. Nuclei were arranged around the peripheral margins of the cytoplasm as dense spherical masses about 1.2/um in diameter.

Merozoites (Figs. 21 - 26 ) Mature macroschizonts (Figs. 21 - 2ifc ) were found to contain tv;o,four and eight macromerozoites. These had a pointed anterior end and a broadly rounded posterior end. Cytoplasm resembled a loose net work with a terminal 151

spherical nucleus. Macromerozoites measured 3.5 Aim - 12 yumXl.6Ami-4/im ( average 5.7/urn X 2.4 Am). Average measurement of the nucleus was 1.2 Aim in diameter. Mature micromerozoites were not found. In one individual ( Fig.23) a mature macroschizont was found to contain four elongate merozoites with a large amount of residual body by their side. Cytoplasm was reticular and light pink in colour while nuclei were deep blue. Macromerozoites ( Pig. 26 ) were also found in lung smear. Two of these lay on two sides of the periphery of macroschizont and measured 10 Aim - 12 Aim X 3.3 Aim - 4 Aim ( average 11.5 Aim X 3.3 Aim ).

Effect of parasites on host cells

Invasion of thiss parasite caused slight linear hypertrophy of infected erythrocytes as shown in the following table.( measurementss in micrometers, Aun )

Ran ge. Ave rage. Length of normal erythrocyte* 12 - 16.5 15 Width of normal erythrocyte. 7,5-11.5 9.8 Length of normal erythrocyte nucleus. 3.3-6.6 5,3 Width of normal erythrocyte nucleus. 2.2-3.8 2.9 Length of infected erythrocyte. 16,3-20.9 18.2 Width of infected erythrocyte. 7-12.2 9.5 FIGS. 13 - 26 . Camera j^cida drawings of the tissue stages of Mepatozoon yusp. (a.). FIGS.13 - 14. Trophozoites in tissue sections of liver. FIG. 15. Blnucleate schizont in tissue sections of liver. FIGS.16 - 20. Immature and mature microschizonts in tissue secticn s of lung. FIGS. 21 - 24. Diimature and mature mncromerozoites in tissue sections of lung. FIG,25. Macromerozoltes in tissue sections of liver. FIG. 26. Macromerozoltes in lung smear. 3. X 1300

EIGURE|; r - 3. Photomicrographs of macromer©zoites of ^' Ifepatozpon n.sp. (a) ,in the sections of 1%'er and limg tissues. 1'. Two macromerozoites in liver section, 2. : I 3. Four macrometozoites;in lung section Eiqht raacrom^rozoites:; in liver section. , k 152

Length of infected erythrocyte nucleus. 5.2-10 7.1 Width of infected erythrocyte nucleus 1.6-3.3 2.2

Nucleus of erythrocyte v/as usually markedly displaced to one side. Sape of hypertrophied infected cells depended on the position of parasite and on displaced host cell nucleus. Host cell nucleus was not seen in fragmented condition.

Movement of parasite

In citrate solution,parasites moved slowly within host cells.

Discussion

Occurrence of schizogony in cells of the liver and lung of the snake host identifies the parasite as a member of the genus Hepatozoon (Miller,1908; Garnham,1950; Mohammed & Man sour, 195%; Btay,l964). Levine et al., (1980) placed the genus Hepatozoon in the Family Hepatozoidae of the sub order Adeleina under the Class Sporozoea. Landau(1973a, b) and Landau ejt al., (1"972)described a Haemoqreqarina sp. in an arthropod and a Hepatozoon sp. in a leech. They 153

reported tissue cysts in the Hepatozoon which are capable of infecting predators. Tissue cysts were not observed in the parasite described here. The parasite under discussion resembles Heoatozoon minchini Garnham (1950) , Heptntozoon rarefaciens Ball et al,, (1967) and Hfepatozoon fusifex Ball et^ &!_•» (1969) in the general pattern of schizogony in tissues of the snake but differs in other morjphological features as v;ell as in the types of tissue schizonts and merozoites. In Hepatozoon minchini^ gametocytes in peripheral blood and schizonts and merozoites in lung sections exhibit only a single morphological type. In Hepatozoon rarefaciens and in Hepatozoon fusifex gametocytes are of one type and three types( depending on the nature of infected ery­ throcytes) respectively. Ball et al./ (1969) considered that more than one species of haemogregarine occurred in Boa constri­ ctor. A comparative study of these species of Hepatozoon with the present parasite is given in table 1 which reveals that the present parasite does not completely resemble any of or any other known species of the genus, and it is therefore considered as a new soecies. 15 4' TABLE 1

Comparative study of Hepatozoon minchinl Gamham,1950; Hepatozoon

rarefaciens Ball ejt ai., (1967); Hepatozoon fusifex Ball et. al^., (1969)

and the present parasite,Hepatozoon n.sp.(a) ( measurements CRongeJ in

micrometers. Aim) .

H. minchinl H.rarefaciens H.fusifex H.n.sp, (a) Gametocytes( Intra-erythrocytic)

One type. One type Three types Two types as per description of gametocytes.

13 - 14 X 3 - 4. 11-18 X 2.5 -9 9,2-17.3 X 2.3-8.1 5.5-13.2 X 15-23 X 4.6-11.5 3.3-9.2 15-18.4 X 4.6-9.2 9.7-16 X 1.4-2.5 Sexual dimorphism Monomorphic. Monomorphic, Trimorphic, Dimorphic; sexes not promi­ sexes not nent. stated.

Development in vertebrate host: In liver In lung tissue. In liver,lung^spleen. In lung,liver, pancreas &. heart tissues.spleen,brain,heart •, " SEkir^ney tissues. ^

Types of tissue schizonts:; One type with Two typess Two types vjith Tv/o types raerozoites with mero2;oites. merozoites. with merozoite

Vector Culex tarsalls. Culex tarsalls Unknown. Unknown. Anopheles albimanus Aedes togoi, . I II .1.1 .1 . II . -^des sirrensis itoblyomma dissimile Site of infection: Peripheral blood. Peripheral Peripheral Peripheral blood. blood blood Vertebrate host. Crotaphopeltis Drymarchon Boa constrictor Ptyas mucosus degeni corals Constrictor constrictor Locality: KavironKavirondd o Gulf of: Colma & Tepic Colima, Mexico Bongaon, Lake Victoria,East Africa. Mexico North jsmerica. West bengax, India. r55

Diagnosis

Family. Hepatozoidae Wenyon,1926. Schizogony in cells of internal organs of verteberate host. Genus. Hepatozoon Miller,1908 Intra-corpuscular ; non-pigmented parasite; schizogony in cells of liver and lung tissues of snake host; gametocytes intra-erythrocytic; sexual dimorphism of gametocytes not prominent. Specific characters. Gametocytes exhibiting two morphologiffial types; Type. I. 5.5/um - 11 Aim X 1.6'"- 3.S/um Type. II. 9,7/um -16 Aim X 1.4^- 2.5 Am Development in vertebrate host(Tissue phase). Trophozoites. 6.4 Aim - 7.7/um X 4.8/um -6.5Afflaf Schizonts aree of two types: Type.I. Macroschizont, (10/um - 13/um X 8/um - 11 Aim) with macromerozoites (3.5/um - 12/um X 1.6 /um - 4 /um) Type. II. Microschizont ( 7.7/um - 11 ;um X 6.5 /Um - 8.8 /um ) . For tthe present the parasite is referred to ^^ Hepatozoon n. sp, (a^), Host. Ptyas mucosus (Linnaeus) Site of infection. Peripheral blood. Liver and lung tissues, Locality : Bongaon,24-Parganas,West Bengal,India. 156

Hepatozoon in Ophidia

Parasite No.2* Hepatozoon" n. sp. (B) ( PIGS. 1 - 16)

Occurrence The parasite was observed in peripheral blood smears of sttriped keel -back snalces ( Amphiesma stol^ta [Linnaeus] ) collected from Chakdah,Hadia/West Bengal,India.

Morphology Parasites in the circulating blood were either intra- or extra-corpuscular. These were in the form of gametocytes which are at various stages of development.

Gametocytes (Figs. 4-14) These were abundant in blood smears and only one morphological type is recognised depending on their size, shape and staining properties. Young individuals (Figs. 4-5) were small, oval or bean-shaped measuring 8.8/um - 9.4/am X 2.2 Aim - 2.4 Aim (average 9/um X 2.3 Aun ). Cytoplasm was homogeneous,non-gra­ nular, non-vacuolated and stained light blue. Nucleus was elonqate or oval ,terminal or sub-teeminal, deep pink in colour,occupying the greater part of cell body. Average measurement of the nucleus was 4.5/um X 2.5 Aim . Cases of double infection of 157

erythrocytes were noticed. Ohe young parasite and a mature parasite could be seen occurring in both sides of host cell nucleus (Fig.5 ). As development proceeded, mature young forms (Pigs. 6 - 7 ) were enclosed in a capsule with a limiting membrane. These became slender and sausages--shaped measuring 11 Ann - 13 Aim X 2.2/um - 4.4 /om (average 12/um X 3.3/um ). Cytoplasm was homogeneous and faintly stained. Nucleus was sub-spherical to oval, sub-central and deep pink in colour. Mature forms(Figs. 8 - 11 ) were long slender and vermicular. These were

capsulated forms measuring 16,5 Aim - 19.8 Aim X 2,5/UIW*3AMI ( average 18/um X 2.7 Aim)* Cytoplasm and nucleus showed similar features as in mature young forms. Average measure­ ment of the nucleus was 4/um X 1,7/um. Double and triple infection of erythrocytes by mature gametocytes were also found. Two to three gametocytes were seen crowding inside host cell which was much distorted (Figs, 9 - 10). Parasites; infecting anucleated cells were also seen and double infection of such case was also observed (Fig.11 ), Ex-capsulation of these gametocytes were encountered in extra-cellular form in blood smears, where parasites were seen to leave capsule. In one case (Fig. 12),the parasite was about to escape,and only a part of its body remained inside capsule. Capsule was colourless,transparent,permiable measuring 17,6 Aim X 5.5 rss

Ex-capsulated extra-cellular gametocytes (Pigs.13 - 14) were narrow longi'.a'*! elongate with tapering ends of which one end was more pointed than the other. These forms measured 17.8 Am - 19.3 Aim X 1.5 Aim - 2.2 Aim { average 18. 4/uni X 1.9 Aim ), Cytoplasm and nucleus showed similar features as in the case of late young forraau Averaqe measurement of the nucleus was 4.5/um X 1.8Aim,

Tissue phase.

No erythrocytic schizogony was seen in peripheral blood smears. Parasite showing various stages of development were detected in sections of liver and lung tissues. Trophozoites (Fig.l ) Early forms were spherical to ovoid measuring 6 Aim - 9 ^im X 4/um - 6.5 Aim ( average 7.2 Aim X 5 /um ), Cytoplasm was homogeneous/non-granular,non-vacu- olated and stained light pink with eosin stain. Nucleus was large, oval and composed of dark-staining chromatin granules which took up blue colour with haematoxylin stain. Average measurement of the nucleus was 2.5/jmK; 2 Aim* Trophozoite (Fig.l) was surrounded by a clear vacuole. It was amoeboid with four cytoplasmic processes and a centr­ ally placed large nucleus which measured 2.2/um in diameter. 159

Sehizont (Fig.2)

Early form contained four nuclei measuring 7.5/Um X 6/umia Cytoplasm was homogeneous> non-granular and stained light pink with eosin. Nucleus was spherical /deep blue with haematoxylin and measured 2 /um in diameter. As the development advanced/two kinds of schizonts were formed. Mature macroschizonts measured 11 Aim X 7.7 Aim^and produced four macromerozoites. Microschizonts (Figs. 15 - 16) were sub-spherical to oval measuring 7.5/um - 16/am X 7.Aim - 10.5 Aini ( average 11 Aim X 8/um )) with sixteen ,thirtysix and sixty or more nuclei. Cytoplasm was reticular,pink in colour and contained dark blue nuclei. Xn some the nuclei were also arranged arotind peripheral margins of schizocyst as dense spherical masses, measuring 1.8 Am in diameter.

Merozoites (Fig.3)

Mature macrosclzonts were found to contain four macromerozoites (Fig. 3). These were with a blunt or somewhat pointed anterior end and a broad poster­ ior end. Macromerozoites measured 5.5 Aim - 8 Aim X 3 Aim - 4.5 Aim (. .average 6.5/um X 3.2 Aim ). Cytoplasm was reticular/non-granular and light pink in colour with eosin. Nucleus was spherical/sub-terminal,compact and stained deep blue with haematoxylin. Average measurement of the nucleus 10 11: 12 13 14 5*193. 13-14. Free gametocytes, FIGURES 1-14. Camera lucida drax-zings of Hepatozoon n.sp.(b) 1. Trophozoite in liver section. 2. Quadrinucleate schizont in liver section. 3. Four macromerozoites in liver section. 4-14, Gametocytes•in erythrocytes. 4 - 6. Developing gametocytes in erythrocytes. 5. Double infection by immature and mature gametocytes 9 - 11. Double infection,triple infection and gameto­ cytes infecting anilcieated erythrocytes. 12. Mature gametocyte escaping from capsule. i&. photc»nic IS of microschizonts of: Hfepatozooi n. at). (b) in the^ sections' of^ lunl reo was 2.5 Aim in diameter. No residual mass was present with macromerozoites. Microschizonts were not fotmd to contain mature micromerozoites.

Effect of parasite on host cells. Invasion of this parasite caused slight linear hypertrophy of infected erythrocytes as shown in the following table ( measurements in micrometers,/mi)

Range. Average, Length of normal erythrocyte. 13.5-17,8 15.7 Width of normal erythrocyte. 8.5-11.5 9,9 Length of normal erythrocyte nucleus. 5.5-6.6 5.7 Width, of normal erythrocyte nucleus, 2.2-4.4 3,2 Length of infected erythrocyte, 16-22.5 19,4 Width of infected erythrocyte.nucleus. 8.8-13.2 11 Length of infected erythrocyte nucleus. 5.6-9 7.2 Width of infected erythrocyte nucleus. 2.2-3.3 2.7

Nucleus of erythrocyte was usually markedly displaced to one side. Shape of hypertrophied infected cells depend on the position of parasite and on displaced host cell nucleus. Host cell nucleus was not seen in fragmented condition. Movement of parasite In citrate solution/ parasites moved slowly within host cells. 1'61

Discussion Developmental stages in lung and liver tissues of the snake host indicates that the parasite belongs to Adelea type of genus Hepatozoon. Parasite under discussion resembles Hepato zoon minchinxGamham (1950), Hepatozoon rarefaciens Ball e^ B.1. , (1967) ,Hepatozoon fusifex Ball et al.,(1969) and Hepatozoon n.sp.(a) in the general pattern of schizogony in tissue of verte­ brate host but differs in other morphological features, as well as in the type of tissue schizonts and merozoites. Erythrocytic forms are smaller in Hepatozoon minchini and tissue schizonts and merozoites are of one kind, Gametocytes of Hepatozoon rarefaciens and Hepatozoon fusifex are different in structure and measurements. Moreover, a capsule is not known in their mature forms. In Hepatozoon n.sp.(a) two morphological types are knov/n. The parasite described in the present study differs from all other known species from snake host on the basis of its morphological characters described above and therefore it appears necessary to consider it as a new species. 162

Diagnosis:

Family. Hepatozoidae Wenyon,1926. Schizogony in cells of internal organs of vertebrate host. Genus. Hepatozoon . Miller,1908. Intra-corpuscular; non-pigmented parasite; gametocytes intra-erythrocytic ; sexual schizogony in cells of liver and lung tissues of snake. Specific characters. Garnetocytes exhilfiiting single morpholo­

gical type; 16.5 Aim S.SAUT^ - 19.8/um X 2.2/um - Si/um* Development in vertebrate host ( Tissue phase) Trophozoites measuring 6 Aim - 9 Aim X 4:/am - 6.5 Aim; Schizonts exhibiting two types; Macroschizont ( 11 Aim X 7.7 ^Aim) with 4 macromerozoites; Microschizont C7,5/urn - 16:/im X 11 Aim - 8 Aim ). For the present parasite is referred to ^'^ Hepatoooon n. sp. (h) •

Host. Amphiesma stolata. (Einnaeus) Site of infection. Peripheral blood.^-iver and lung tissue Locality:: Chakdah,Hadia,West Bengal, India. m3

Hepatozoon in Ophidia. Parasite No.3 Hepatozoon n.sp. (c) (FXGS.. 1-11)

Occurrence The parasite was found in peripheral blood smears of a cobra,Naja naja (Linnaeus)_ collected from Bamipur, 24-Parganas,West Bengal,India.

Morphology Parasite in blood films was in the form of intra- erythrocytic gametocytes which were at various stages of development. No extra-corpuscular form of parasite was observed. Gametocytes (Figs. 1-5) These were abundant in blood smears. Two distinct morphological types were detected depending on their size,shape and staining properties. These two types might represent t\^o different sexes,which could not however, be conclusively distinguished. Type. I, Early forms of these gametocytes were not seen. Young forms (Figs. 1 -2) were encapsulated,elongate, oval or slightly broad with two globular ends. These measu­ red 12/um - 13.8/um X 2,7 yum - 3.3/urn ( average 12.8/um X 2.9 im

/um),. Cytoplasm was homogeneous,non-granular and stamhed light blue in colour. Nucleus was compact,sub-spherical deep pink and with an average measurement of 3.2/um X 2/jm. Mature forms were slender,encapsulated and with a blunt tapering end at one pole with a broad globular end at the other. These measured 14.8/um - 16.5/um X 2.6/um -3.4/um Cytoplasm shov/ed similar features like late young forms. Nucleus was central,compact,oval,deep pink in colour and the average measurement was 3.6/um X 2.4/um.

Type. ir.

These (Figs.4-5) were oval,capsulated measuring 12.2/um - 14/um X 2.2/um - 2.5/um( average 13.2/um X 2.3/um). Inside capsule,parasites were found to contain polar caps at both ends which took up a deep blue colour and fitted over parasite as a cup. Polar caps were 2.2/um in length and remained in oblique or slanting fashion. Cytoplasm was homogeneous,non-granular and basophilic. Nucleus was light pink,non-compact,elongate or stretched with dispersed chromatin granules occupying maximum part of cell body. Average measurement of the nucleus was 7.5/um X 1.3/am. Ii65

Tissue phase.

N5 intra-erythrocytic schizonts were ever encountered in peripheral blood smears. Parasites showing various stages of asexual reproduction in sections of liver were found. Mature schizonts were also observed in blood

smears. Trophozoites (Pigs. 7-8)

Early trophozoite (Eig.8 ) was ovoid and measured 5/um X 3.8/jm» Cytoplasm was homogeneous,non­

granular, non-vacuolated and took up light eosin stain.

Nucleus was round or oval and composed of dark blue chromatin

granules in haematoxylin stain. Nucleus was round or oval.

Trophozoites were surrounded by a vacuole. In one indivi­

dual (Fig. 7 )) the parasite was amoeboid or irregular in shape

and measured 9/um X 4.6/um. Nucleus was large,central,

spherical and measured 3.2/um in diameter.

Schizonts. (Figs. 9 - 12)

Development of parasite inside liver

indicated two types of schizonts. Macroschizont (Fig.9)

measured 13.2/um X 12/um containing two macromerozoites.

Microschizonts (Figs. 10 - 12). were sub-spherical measuring

11.5/im - 16.5/um X 10 ^,um - 14.5/um C average 14/um X 12/um )

with 11 - 12 nuclei. Cytoplasm was reticular and took up

light eosin stain containing scattered nuclei within it. FIGURES.1 -12. Camera lucida drawings of Hepatozoon n.sp. (c). Figs. 1 - 3. Gametocytes Type. I. Figs. 4-5. Gametocytes Type.ir. Fig.6.Normal erythrocyte. Pigs. 7-8, Trophozoites in liver sections. Fig. 9. Macromerozoites. Figs. 10 - 12. Microschizonts. 1. X 135w

FIGURE 1." Photomicrograph of microschizont off Hfepatozoon n.sp. ( c ); in liver smears. rea

Nuclei were arranged around peripheral margins of mature' schizocyst as dense spherical masses which stained dark blue with haematoxylin stain. Microschizont;; (Pig. 16) was also found in peripheral blood smears and measured 25/um X 17 /um. It contained 21 nuclei which took up dark pink colour in Leishraan's stain.

Merozoite (Fig. 9)

Mature macromerozoites ( Fig. 9) were two in number lying side by side. These were 8.5 Aim -9/um X 3/lim - 3.5/um (average 8.7/um X 3.2 Aim ). These were with a blunt or somewhat pointed anterior end and a broad posterior end. Cytoplasm was homogeneous^non-granular: and stained light pink with eosin stain. Nucleus v/as spherical,compact and sub-terminal. No residual body was detected. Micromerozoites were not observed.

Effect of parasites on host cells. Invasion of this parasite caused slight linear hypertrophy of infected erythrocytes as shown in the follov/ing table ( measurements in micrometers, /um) .

Range. Average. Length of normal erythrocyte. 14.3-17.7 16 Width of normal erythrocyte. 8.8- 11 9.9 167

Length of normal erythrocyte nucleus. 5.5-7.77 6.4 Width of normal erythrocyte nucleus. 2.7-4.4 3.5 Length of infected ;'. erythrocyte. 15.5-22 19.6 Width of infected erythrocyte. 7.7 - 13 9.6 Length of infe-cted erythrocytic nucleus 5.2-8.2 7.3 Width of infected erythrocyte nucleus?. 1.9-2.3 2.1

Nucleus of erythrocyte was usually markedly displaced to one side. Shape of hypertrophied infected cells depended on the position of parasite and on displaced host cell nucleus. Htist cell nucleus was not seen in fragmented condition.

Movement of Parasite. In citrate solution, parasite moved slowly within host cells. 168 Discussion Schizogony in the liver cells of cobra has placed the present parasite under genus Hepatozoon« The parasite resembles Hepatozoon minchini Garnham (1950), Hepatozoon rarefaciens Ballet a,l., (1967) , Hepatozoon fusifex Ball et_ al., (1969), Hepatozoon n.sp.(a^) and Hepatozoon n.sp.(b) in general pattern of schizogony in tissue of the snake host, but differs in other morphological features>as v;ell as in the type of tissue schizonts and merozoites. In Hepatozoon minchini , gametocytes in peripheral blood and schizonts and merozoites in the lunig sections exhibit single morphological type. In Hepatozoon rarefaciens and Hepatozoon fusifex the gametocytes are very distinct and different from the parasite described here. In Hepatozoon n.sp. (_a) and ih) the gametocytes in peripheral blood and the tissue schizonts and merozoi­ tes are quite distinct and different from the present case. A comparative study of the present three new species is given ( page no. 170 ) which indicates that these species are different frsm each other in their erythrocY^ic forms as well aaifltissue forms. Thus,the parasite described here is considered a new addition to science a^^d named here as Hepatozoon n.sp,(£)

Diagnosis Family Hepatozoidae Vtenyon,1926, Schizogony in cells of internal organs of vertebrate host. 169

Genus Hepatozoon Miller, 1908. Intra-corpuscular; tissue schizonts in the cells of liver of snake host} Se:-cual dimorphism not prominent . Specific characters In the present study, gametocytes exhibiting tv/o morphological types. Type I. 12/um - 16 .5 Aim X 2.6/um -3.4 A^n* Type II. 12.2 Aim - 14Aiin X 2.2/m - 2.5/urn. Development in vertebrate host i Trophozoite. 5/um - 9/um X 3.8/um - 4.6/jm Macroschizont. 13.2/um X 12 /um:/; with mnrozo- ites 8.5 Aim - 9/om X 3/wn - 3.5 Aom. Microschizonf* 11.5 ATO -25 Aim X 10 Aim - 17/am.

For the present,the parasite is referred to as Hepatozoon n.sp. (c)

Host. Naja naja (Linnaeus)' Site of infection. Peripheral blood S;-liver Locality: Baruipur, 24-Parganas, West Bengal,India. 170 Comparative study of Hepatozoon n. sp • (a),, (b). & ic) in snakes; collected in some parts of West Bengal (measurements in micrometers) Hepatozoon n.sp. (_a) Hepatozoon n.sp. (b) Hepatozoon n .sp. {c)

Blood forms

Gametocytes Tv/o types One type Two types Type I 5.5 - 13.2 X l,e - 3.8 16.5 - 19.8 X 2.5-3 12-16.5 X2.6-3.4 Type II 9.7 - 16 X 1.4 - 2.5 12.2 -14 X 2.2 -2.

Sexual dimorphism Dimorphic; saxes could Monomorphic, Dimorphic;sexes not be conclusively distinguished. could not be conclusive ly distinguished. Development in vertebrate host In the liver & lung tissues. In the liver & In the liver tissue. lunq tissues.

Trophozoite Trophozoite; Macroschizont Trophozoite; macro- macroschizont with with macromerozoites; schizont with macromerozoites; macromerozoites; Microschizont. microschizont. microschizont .

Invertebrate host. Unknown, Unknoim. Unkno;vn, Site of infection Peripheral blood,liver Peripheral blood. Peripheral blood, &': lung tissues. liver & lung tissues. Si liver tissue. Vertebrate host Ptyas mucosus(Linnaeus) itophiesma stolata(Linnaeus) Naja naja (Linnaeus) Loc al ity: Bongaon,24-Parganas Chakdah,Nadia,WestBengal. Baruipur,24-Par- West Bengal,India. India. ganas,West Bengal, India. 171:

CI-iECK-LIST OF Hepatozoon IN INDIMI SNSCES (REPTILIA)

( * indicates new species described in this thesis)

Family. Hepatozoidae Wenyon,1926. Genus.. Hepatozoon Miller^ 1908.

!• Hepatozoon sp. Sinha/1980. In the blood of amphiesma stolata#collected from Chakdah, lsradia,West Bengal. * 2. Hepatozoon n.sp.(a) In the blood of Ptyas mucosus. collected from Bongaon,24-Parganas~;, West Bengal, * 3. Hepatozoon n.sp. (b^) In the blood of Anphiesma stolata ,collected from Cha3cdah^ Nadia, West Bengal. * 4. Hepatozoon: n.sp. (c) In the blood of Naja naja , collected from Baruipur,24- Parganas, West Bengal.