SCHELLACKIA CALOTESI N . SP. FROM AGAMID LIZARDS OF THE GENUS CALOTES IN THAILAND

SCHELLACKIA CALOTESI N . SP. FROM AGAMID LIZARDS OF THE GENUS CALOTES IN THAILAND

FINKELMAN S* & PAPERNA I.*

S u m m a r y :

Schellackia calolesi n. sp. is described from the Thai agamids Calotes mystaceus and C. versicolor. Schellackia-type sporozoites were recovered from blood and liver of one C. versicolor from Kon Kaen North-East Thailand and two C. mystaceus from Chiang Mai, North Thailand. Specimens of both species were fed on sporozoite infected blood, of these only one C. mystaceus developed endogenous infection in the anterior intestine.

Description, from histological material includes early and dividing meronts, micro and macrogamonts and non sporulated oocysts.

KEY-WORDS : Schellackia calotesi, meronts, microgamonts, macrogamonts, oocysts sporozoites, gut, liver, blood, Calotes mystaceus, Calotes versicolor, Thailand.

Résumé : SCHELLACKIA CALOTESI n. sp. CHEZ LE LÉZARD DU GENRE CALOTES EN THAÏLANDE

Schellackia caloseti n. sp. est décrit chez les lézards thaïlandais Calotes mystaceus et C. versicolor. Des sporozoïtes de type Schellackia ont été retrouvés dans le sang et le foie d'un C. versicolor de la région de Kon Kaen, au nord-est de la Thaïlande, et de deux C. mystaceus de Chiang Mai, en Thaïlande du nord. Des spécimens de chacune de ces deux espèces nourris de sang infesté de sporozoïtes, seul un C.

mystaceus a développé une infection au niveau de l'intestin antérieur. La description à partir de coupes histologiques concerne des mérontes jeunes et en cours de division, des micro- et macrogamontes et des oocystes non sporulés.

MOTS CLES : Schellackia caloseti, mérontes, microgamontes, macrogamontes, oocystes, sporozoïtes, intestin, foie, sang, Calotes mystaceus, Calotes versicolor, Thaïlande.

INTRODUCTION

T

h e g e n u s Schellackia c o m p r i s e s nine hetero- g e n e o u s s p e c i e s o f eimerian coccidia, parasites o f either saurian reptiles or anurans transmitted by h e m a t o p h a g o u s Acarina or insects (Klein et al., 1 9 8 8 ; Bristovetzki & Paperna, 1 9 9 0 ) . T h e few k n o w n s p e c i e s have n o n e t h e l e s s b e e n reported so far from all continents e x c e p t Australia. T h e presently described n e w s p e c i e s is from a Thai agamid lizard.

MATERIALS AND METHODS

T

h r e e Calotes versicolor and o n e C. mystaceus w e r e c a p t u r e d in T h a i l a n d near K o n K a e n (Northeastern r e g i o n ) and 12 C. versicolor and 34 C. mystaceus from Chiang Mai (Northern region) ( 1 2 and 34, respectively). Films w e r e prepared from b l o o d o b t a i n e d by clipping the tip o f the lizard's tail. Smears w e r e prepared from the gut and liver. Prepared films and smears w e r e air-dried fixed in absolute methanol

* Department of Animal Sciences, Faculty of Agriculture of the Hebrew University of Jerusalem, Rehovot 76100, Israel.

Correspondence: llan Paperna. Fax: 972 8 465763.

E-mail:Paperna@agri.huji.ac.il

and stained in Giemsa ( 1 / 1 0 diluted in phosphate buffer pH 7 . 4 ) for o n e hour. T w o apparently uninfected spe- cimens o f each species were fed on blood and liver o f a sacrificed infected C. versicolor from Kon Kaen and sacrificed after 12 days. T h e gut and the liver were fixed in neutral buffered Formalin for histology. Fixed tissue, after dehydration in graded ethanols, was e m b e d d e d in glycol-methacrylate medium (GMA o f Agar Comp., UK).

Sections, 3-4 Lim were cut with a glass knife in a Sorval J B 4 microtome and stained with Meyer's haemalum-

eosin (MH-E). Descriptions o f all developmental stages were made from histological sections and refer to cross sections. All measurements in micrometers (μm).

RESULTS

O

f all e x a m i n e d lizards sporozoites w e r e r e c o ­ vered only in o n e C. versicolor from K o n Kaen, and in t w o C. mystaceus from Chiang Mai. In the first, sacrificed for histology, sporozoites o c c u r r e d in the liver as well as in the b l o o d , but n o stages w e r e found in the intestine. O f the two C. mys­

taceus and two C. versicolor fed on infected b l o o d and liver, only o n e s p e c i m e n o f the first s p e c i e s d e v e l o p e d an e n d o g e n o u s infection.

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F I N K E L M A N S . & P A P E R N A I

Fig. 1. - A, B: Young meronts.

C: Young microgamont. D:

Premature microgamont. E:

M a t u r e m i c r o g a m o n t . F:

Y o u n g m a c r o g a m o n t . G: Mature macrogamont. H:

Zygote/young oocyst. I: Free sporozoite.

Abbreviations: A: amylopectin granules; dwf: disaggregating wall-forming bodies; L: lipid vacuole ; Mi: microgametes ; Nu: nuclei; nu: nucleolus ; R.

refractile body ; rn: residual nuclei; wf: type 1 wall-for­

ming bodies ; Wf: type 2 wall- forming bodies.

SCHELLACKIA CALOTES1 N. SP.

T y p e host: Calotes mystaceus.

Locality: Chiang Mai, Thailand.

O t h e r hosts and o c c u r r e n c e : Sporozoites w e r e traced in the b l o o d o f two C. mystaceus from Chiang Mai and o n e C. versicolor from K o n Kaen, Thailand.

DESCRIPTION

E n d o g e n o u s stages (Fig. 1) o c c u r r e d in the mucosal epithelial layer o f the anterior gut (Figs. 2 - 6 ) . All stages e x c e p t mature oocysts w e r e lodged in an e x p a n d e d parasitophorous v a c u o l e ( P V ) . Although most infected cells hosted o n e parasite, a few h o w e v e r contained two (Figs. 2, 4 ) or even three macrogamonts or both macro- g a m o n t s and m i c r o g a m o n t s . Meronts had a d e n s e

cytoplasm with very few, or n o inclusions o r v a c u o l e s (Fig. 1A). Nuclei c o n t a i n e d a very distinct nucleolus.

Meronts ranged from 5.6 x 2.8 (with two nuclei, Fig. 1A) to 8 . 4 x 8 . 4 (with six nuclei, Fig. 1B ) , their PVs sizes ranged from 7 x 4 . 2 to 1 1 . 2 x 7 . 7 . Premature meronts w e r e s e e n with up to eight nuclei per section and divi­

ding meronts s h o w e d up to ten emerging m e r o z o i t e s (Figs. 2, 3, 5).

Young microgamonts with eight to nine nuclei were 7- 9 x 6 . 2 - 8 . 4 in size, and were located in a 9 . 8 - 1 2 . 6 x 8 . 4 - 11.2 PV (Figs. 1C). T h e cytoplasm was lucent to foamy and the nuclei had a h o m o g e n e o u s consistency (Figs. 2, 3). Premature and mature microgamonts, 11.2-14.0 x 6.2- 8.0 in size (Figs. I D , E ) , were lodged in a 16.8-18.2 x 9.8- 14.0 PV. Toward the completion o f differentiation the microgamont cytoplasm b e c a m e increasingly vacuolated.

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SCHELLACKIA CALOTESI N. SP. FROM AGAMID LIZARDS OF THE GENUS CALOTES IN THAILAND

Figs. 2 to 6. - View of histo- logical cross sections in the anterior portion of the intes- tine ; double infections in host cells shown in Fig. 2 ii & z, and Fig. 4 a ¿4 z and o & o ; arrows denote the limits of the basal membrane (Fig. 6 ) ; GMA-embedded, glass knife/

JB4-sectioned, MH-E stained (Figs. 2, 3, 5, 6 x 6 5 0 ; 4 x 8 3 0 ) .

Figs. 7-8. - Fig. 7. Sporozoites in the liver, adjacent to macro- phages ( m ) and melanoma- crophages (M), smear, Giemsa stained ( x 500). Fig. 8. Sporo- zoites in the blood erythro- cytes (arrows), Giemsa-stained blood film ( x 1100).

Abbreviations: a: young macro- gamont ; E: epithelium ; i:

mature microgamont; ii: pre- mature microgamont; Lp: la- mina propria; o: oocysts; s: pre- mature meronts; z: zygote.

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FINKELMAN S. & PAPERNA I.

Parts o f the cytoplasm seemingly disaggregated, while retaining a dense cytoplasm where a few residual or non- differentiated nuclei remained. Microgametes flagella w e r e conspicuously long (Figs. 3, 4 ) .

M a c r o g a m o n t s had a characteristically large vesicular nucleus with a large, deep-staining nucleolus (Fig. 4 ) . Y o u n g macrogamonts, 7.0-8.4 x 7.0-8.0 in size (Fig. 1F), had a h o m o g e n o u s cytoplasm, with large v a c u o l e s (apparently amylopectin granules) appearing gradually with maturation (Figs. I F , 3, 4 ) . In apparently mature 7 . 0 - 9 . 8 x 8 . 4 - 9 . 0 m a c r o g a m o n t s m o r e than half o f the cytoplasm volume was filled with amylopectin granules and wall-forming b o d i e s w e r e visible (Fig. 1 G ) . B o t h y o u n g and mature forms w e r e lodged in 9 - 8 - 1 0 . 5 x 8 . 4 - 9.8 PVs.

Zygotes or early oocysts r e a c h e d 1 5 . 4 x 9 . 8 in size, and w e r e lodged in a PV reaching up to 19.6 x 11.2 in size (Figs. 1H, 2, 4 ) . T h e y w e r e densely filled with amylo­

p e c t i n granules a n d in their nuclei, t h e n u c l e o l u s b e c a m e less c o n s p i c u o u s or disappeared. Wall-for­

ming bodies, c o n s p i c u o u s (type 2 ) and minute (type 1) occurred in the cytoplasm, the first concentrated mainly along the o o c y s t wall (Fig. 1 G ) .

Mature o o c y s t s w e r e almost round ( 9 . 8 - 1 0 . 5 x 9-0-9.8), lodged in 1 1 . 2 - 1 1 . 8 x 9 . 8 - 1 0 . 5 PVs, and d e n s e l y filled with a m y l o p e c t i n g r a n u l e s . T h e c e n t r a l l y l o c a t e d nucleus had an irregular perimeter and w a s h o m o g e - nously d e n s e . Wall-forming b o d i e s , or their disaggre­

gated product, w e r e c o n c e n t r a t e d b e n e a t h the rims o f t h e o o c y s t w a l l ( F i g s . 1H, 4, 5 ) . M a t u r e o o c y s t s o c c u r r e d in b o t h the epithelial layer (Figs. 2, 4 ) , and lamina propria (Figs. 5 , 6 ) ; s o m e a p p e a r e d to b e pres­

sing the basal m e m b r a n e into the lamina propria layer (Figs. 4, 5 ) . None o f these oocysts w e r e sporulating, and there w e r e n o free sporozoites. T h e latter w e r e found in another, naturally infected s p e c i m e n , in the liver, next or inside m e l a n o m a c r o p h a g e s (Fig. 7 ) . In the s a m e host, all oocysts had disappeared from the intes­

tine, and in the b l o o d only, erythrocytes w e r e found infected (Fig. 8 ) . S p o r o z o i t e s w e r e 8 . 4 - 1 0 . 5 x 2 . 6 - 5 ; their nucleus was in a s o m e w h a t anterior position and the two refractile b o d i e s w e r e located in the post- nuclear z o n e (Fig. I I ) .

DISCUSSION

S

p e c i e s o f Schellackia demonstrate limited inter­

specific morphological diversity, and the varia­

bility in e a c h stage's dimensions ( a d d e d to the inevitable variation due to p r o c e s s i n g ) is sufficiently large to result in overlapping size ranges a m o n g the s a m e s t a g e s o f different s p e c i e s , e v e n o f w a l l e d oocysts prior to sporulation or waiting intra-erythrocytic sporozoites. H o w e v e r , s p e c i e s may b e otherwise dis­

tinguished according to the particular site at w h i c h

m a c r o g a m o n t fertilization and sporozoites formation occurs, e.g. either in the lamina propria or in the gut epithelial cells, and the sporozoites location in cells and tissues (Lainson et al, 1 9 7 6 ; Klein etal, 1 9 8 8 ; Paperna

& Finkelman,

1996).

Discrimination according to these criteria, restricts the n u m b e r o f s p e c i e s similar to the presently described o n e to the t w o s p e c i e s infecting agamid lizards, n a m e l y S. agamae ( b y Rogier, 1 9 7 4 ) and S. cf. agamae ( b y Bristovetzki & Paperna, 1 9 9 0 ) . High host specificity a m o n g Schellackia spp. has b e e n suggested (Lainson et al, 1 9 7 6 ) and recently has b e e n experimentally also demonstrated, at least at the family level for S. occidentalism S. golvani (Klein et al, 1 9 8 8 ) and S. cf. agamae (Bristovetzki & Paperna, 1 9 9 0 ) . PVs o f all developing stages o f presently described spe­

cies, e x c e p t mature oocysts, w e r e relatively larger than the PVs o f other s p e c i e s . T h e flagella o f the microga­

metes w e r e particularly long. Unlike in the other other s p e c i e s from agamids, sporozoites w e r e not found in circulatory leucocytes. O n the other hand, sizes o f all stages o f Schellackia from Agama spp. (Rogier, 1 9 7 7 ; Bristovetzky & Paperna, 1 9 9 0 ; Paperna, 1 9 9 2 ) and o f S. calotesi overlap. Nevertheless, difference in the host g e n u s (Agama vs. Calotes) and geographical origin (Africa and Near East vs. T h a i l a n d ) s e e m s to b e satis­

factory arguments for specific distinction.

REFERENCES

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K L E I N T.A., Y O U N G E . C . , G R E I N G E R E . C . , T E L F O R D S.R. Jr &

B U T L E R J.F. Development and experimental transmission of Schellackia golvani and Schellackia occidentalis by inges­

tion of infected blood-feeding arthropods. Int. J. Parasitai., 1988, 18, 259-267.

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mission by Culexpipiensfatigans. Parasitai, 1976, 72, 225- 243.

P A P E R N A I. Ultrastructural studies on oocysts, sporulation and sporozoites of Schellackia cf. agamae from the intestine of the starred lizard Agama stellio. Int. J. Parasitai, 1992, 22, 361-368.

PAPERNA I. & FINKELMAN S. Schellackiaptyodactyli sp. nov. of the fanfooted gecko Ptyodactylus hasselquistii from the rift escarpment of the lower Jordan Valley. Folia Parasitai, 1996, 43, 161-172.

ROGIER E. Description et cycle biologique de Schellackia agamae (Laveran et Petitt, 1909), Lankesterellidae parasite d'agames de République Centre-Africaine. Protistol, 1974,

13, 9-13.

Reçu le 10 juin 1997 Accepté le 19 juillet 1997

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