HEPATOZOON KISRAE N. SP. INFECTING THE LIZARD AGAMA STELUO
IS TRANSMITTED BY THE TICK HYALOMMA CF. AEGYPTIUM
PAPERNA E*. KREMER-MECABELL T.* & FINKELMAN S.*
S u m m a r y :
Hepatozoon kisrae n. sp. was found infecting a starred lizard at a site in southeastern Samaria, Palestine. These lizards were also hosts to the ixodid tick Hyolomma cf. aegyptium, which was demonstrated to be the vector of this hemogregarine. Hepatozoon and tick infections occurred in lizards within a very restricted locality; at a second site, nearby, ticks occurred without Hepatozoon infection. Micro- and macromeronts occurred mainly in the lungs, while cyst-like merogonic stages, mainly dizoic, occurred in the liver. Mature intraerythrocytic gametocytes were stout and encapsulated. Development from oocysts to sporocysts took place in the tick hemocoel, and was examined by
transmission electron microscopy. Lizards were successfully infected when fed on sporocyst-infected ticks or viscera of infected lizards.
Ticks become infected when fed on infected lizards; sporogony was complete when the ticks reached adult stage, over 4 0 days after initial attachment.
KEY WORDS : Hepatozoon kisrae n. sp., Agama stellio, Hyalomma cf.
aegyptium, development, transmission, sporogony, ultrastructure.
Résumé : HEPATOZOON KISRAE N. SP., PARASITE DU LÉZARD AGAMA STELLIO EST TRANSMIS PAR LA T I Q U E HYALOMMA CF. AEGYPTIUM Hepatozoon kisrae n. sp. a été découvert chez Agama stellio dans une localité du sud-est de la Samarie (Palestine). Ces Lézards sont égalemant les hôtes de Hyalomma cf. aegyptium. Il est démontré que cet Ixodidé est le vecteur de l'Hémogrégarine. Le cycle ne s'effectue que dans une zone précise : les Tiques d'une région voisine sont indemnes. Les micro et macromérontes se trouvent essentiellement dans les poumons, alors que les kystes (la plupart contenant deux cystozoïtes) siègent dans le foie. Les gamétocytes intraérythrocytaires mûrs, de forme trapue, sont encapsulés. Le développement depuis l'oocyste jusqu'au sporocyste, qui a lieu dans l'hémocéle de la Tique, a été étudié en microscopìe électronique. Les Lézards s'infectent par ingestion, soit de Tiques infectées de sporocystes, soit de viscères de Lézards infectés. Les Tiques s'infectent par repas sur Lézards infectés ; la sporogonie est achevée 40 jours après la fixation de la nymphe, lorque le stade adulte est atteint.
MOTS CLES : Hepatozoon kisrae n. sp., Agama stellio, Hyalomma cf.
aegyptium, développement, transmission, sporogonie, ultrastructure.
INTRODUCTION
T
icks and mites have b e e n shown to vector Hepa- tozoon o f mammalian (Miller, 1 9 0 8 ; Hoogstraal, 1961; Furman, 1 9 6 6 ; M a t h e w et al, 1 9 9 9 ) and avian hosts ( B e n n e t et al, 1 9 9 2 ) ; there is, h o w e v e r , only o n e report o f reptile-host Hepatozoon s p o r o g o n i c d e v e l o p m e n t in a tick (in Amblyoma dissimilae, Ball et al., 1 9 6 9 ) . H e m o g r e g a r i n e s o f the g e n u s Hemolivia, infecting toads (Bufo marinus), lizards (Tiliqua rugosa) and land tortoises ( T e s t u d o graecd) are tick-transmitted, and differ considerably in their course o f d e v e l o p m e n t in their v e c t o r from s p e c i e s o f Hepatozoon infecting reptiles transmitted via insects, notably mosquitoes. In the first, in the tick host, oocysts remain in the gut cells to yield a p r o g e n y o f mobile sporokinetes, w h i c h re- enter the gut tissue to p r o c e e d s p o r o g e n e s i s (Petit et al., 1990; Smallridge & Paperna, 1997; Landau & Paperna,* Department of Animal Sciences, Faculty of Agricultural, food and environmental sciences, The Hebrew University of Jerusalem, Rehovot 76-100 Israel.
Correspondence: Paperna I.
Tel.: 972 8 9489945 - Fax: 972 8 9465763.
E-mail: paperna@agri.huji.ac.il
1997; Smallridge & Paperna, 2 0 0 0 a,b). In the latter, developing in dipteran insects (predominantly m o s - quitoes), oocysts e s c a p e into the h e m o c o e l , sporocysts d e v e l o p and remain to sporulate within the oocyst b o d y (Lowichick et al, 1993)- Hepatozoon o f mam- malian and avian hosts, unergo similar d e v e l o p m e n t into sporocyst loaded oocysts in their vector's h e m o - c o e l ( B e n n e t et al, 1992; Mathew et al., 1 9 9 9 ) . A few s p e c i e s o f n a m e d Hepatozoon s p e c i e s h a v e b e e n reported to b e transmitted via mites (Lewis & Wagner, 1964; Allison & Desser, 1981), their potential affiliation, h o w e v e r , with the mite-transmitted hemogregarinid genus Karyolysus (Reichenow, 1921; Svahn, 1975) needs to b e r e - e x a m i n e d .
T h e starred lizard, Agama stellio, is widespread throu- ghout the Middle East (Haas, 1 9 5 1 ) . D e s s e r & Y e k u - tiel ( 1 9 8 6 - 1 9 8 7 ) e x a m i n e d b l o o d smears from starred lizards c o l l e c t e d from different localities in Israel and Palestine and found two morphologically distinct types o f undescribed hemogregarines, stout and elongate.
T h e e l o n g a t e h e m o g r e g a r i n e is an as yet undescribed s p e c i e o f Hepatozoon (vide sp. A), w h i c h was trans- mitted experimentally via m o s q u i t o e s (Culexpipiens, Finkelman & Paperna, unpublished).
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/200209117
PAPERNA I., KREMER-MECABELL T. & FINKELMAN S.
In this communication w e describe the species forming the stout gametocytes, Hepatozoon kisraen. sp., which infects t h e starred lizard at sites in s o u t h e a s t e r n Samaria, Palestine. T h e s e lizards are also the hosts to an ixodid tick Hyalomma cf. aegyptium, w h i c h is demonstrated to vector this hemogregarine.
MATERIALS AND METHODS
S
tarred lizards (A. stellio) (n = 2 7 ) were captured betw e e n S e p t e m b e r 2 3 , 1999 to August 24, 2000 on the southeastern slopes o f the Samarian mountains in Palestine ( 1 9 7 8 edition m a p o f Israel grids: 155-170 N / 1 8 2 - 1 8 7 E ) , at altitudes o f 5 5 0 to 7 0 0 m a b o v e sea level, in a semiarid Mediterranean habitat (~ 3 5 0 m m annual rainfall) consisting o f rocky areas and terrace- f e n c e d olive groves. T h e collection sites w e r e the fol
lowing: a ) a stone-fenced olive grove, < 1,000 m2 in area ("Kisra", grid 1 6 6 N / 1 8 4 E ) ; b ) its adjoining grounds, at a perimeter o f 5 0 0 m ("near Kisra"); c ) at roadsides along two km on Akraba-Kisra road; d ) a site situated 13 k m d o w n this road to the south ("Roman c a m p " ) and e ) t w o localities situated three and five km o n the road branching eastwards from Kisra (sites c-e are listed as "elsewhere" in table I ) .
B l o o d smears w e r e obtained by clipping the lizard's t o e tip. Ticks w e r e o p e n e d by incision and their vis
ceral contents w e r e e x a m i n e d by direct light micro
s c o p y and from stained smears. Prepared b l o o d films and smears w e r e air-dried, fixed in absolute methyl a l c o h o l and stained for o n e hour in G i e m s a ( 1 0 % in p h o s p h a t e buffer pH 7 . 4 ) . Levels o f p a r a s i t a e m i a defined as: ± light (< 0.5 % ) , + low (0.5-3 % ) . ++ mode
rate ( 3 - 9 % ) , +++ high (> 9 % ) w e r e determined from c o u n t e d 5 0 fields at x 1,000 magnification (~ 4 0 0 ery
throcytes per field).
From moribund, euthanized and freshly dead lizards, tissue samples o f the liver, lungs and sometimes spleen w e r e fixed for histological examination in 10 % neu
tral-buffered formalin. After dehydration in graded ethanols, the tissues w e r e e m b e d d e d in glycol-metha- crylate medium (GMA o f Agar Comp., U K ) . Sections, 3 pm thick, w e r e cut with a glass knife o n a Sorval J B 4 m i c r o t o m e and stained with either Meyer's hae-
malum-eosin or, after a 2 0 min incubation in B o u i n ' s solution and a rinse with water and 7 0 % ethanol, with 10 % G i e m s a , in p h o s p h a t e buffer pH 7.4, for o n e hour.
Captive lizards w e r e kept, e a c h in a glass terrarium 5 0 x 3 0 x 3 0 c m in size under c o n t i n u o u s illumination at room temperature o f 25-31° C and w e r e fed on meal
w o r m s s u p p l e m e n t e d by c h o p p e d c h i c k e n liver and meat. Experimental infection o f lizards was carried out 1) through their feeding o n sporocysts dissected from naturally and laboratory infected ticks (H. cf. aegyp
tium); 2 ) by feeding on b l o o d clots and viscera (mainly liver) o f euthanized infected lizards. T o g e t h e r with the starred lizards, two g e c k o e s (Ptyodactylus basselquistii) w e r e also fed o n viscera o f infected ticks.
Infection-free lizards w e r e obtained from habitats other than the e n z o o t i c location, and c h e c k e d over t w o w e e k period to verify the a b s e n c e o f infection. Visceral contents o f the ticks: males, semi-engorged adult stages and nymphs released by small incision, w e r e e x a m i n e d directly a n d / o r from air-dried, methanol-fixed, Giemsa- stained smears. Positive ticks w e r e used either for an ultrastructural study, or to infect lizards via fee
ding.
E n g o r g e d female ticks r e m o v e d from captured lizards w e r e kept in individual vials for oviposition, in slightly m o i s t e n e d c h a m b e r s (~ 7 0 - 9 0 % RH) at ambient r o o m temperature o f 25-31° C. Laid eggs w e r e incubated, and e n g o r g e d larvae and n y m p h s w e r e allowed to molt in vials under s a m e conditions o f temperature and humi
dity. Hungry larvae, n y m p h s and adults w e r e stored in a 16° C incubator. Lizards, left free or c a g e d in a n e t - b o x , w e r e e x p o s e d to larvae or nymphs in contai
ners with the rims o f their tops aligned by sticky bands.
For transmission electron m i c r o s c o p i c ( T E M ) e x a m i nation, ticks w e r e immersed in 2.5 % glutaraldehyde in cacodylate buffer (0.1 M, pH 7 . 4 ) , tick a b d o m e n was then s e c t i o n e d at the anterior and posterior extremi
ties, and either left that w a y or followed by progres
sive separation o f the digestive tract from the chitinous e n v e l o p e o f the tick. T h e tick material left in the s a m e fixative for 24 h at 4° C, w a s then rinsed in 0.1 M c a c o dylate buffer and postfixed in 1 % osmium tetroxide in the s a m e buffer for o n e hour. After rinsing in the
Kisra Near Kisra Elsewhere
Localities (site a) (site b) (sites c-e)
Total no lizards examined 10 2 15
No. lizards infested with ticks 7 1 4
No. infected with H. kisrae 9 0 0
No. infected with Hepatozoon sp. A 0 0 3
No. lizards with Hepatozoon-infected ticks 6 0 0
Table I. - Summary of numbers of starred lizards examined from localities in Samaria, found infected by Hepatozoon spp. and found infested by Hepatozoon-infected and non-infected ticks.
TICK TRANSMITTED HEPATOZOON OF AGAMA
buffer, the material was dehydrated in graded ethanols and e m b e d d e d in Agar 1 0 0 m e d i u m (Agar Scientific, Ltd., U K ) . Thin sections, cut o n a Reichert Ultracut m i c r o t o m e with a d i a m o n d knife w e r e stained o n grids with uranyl acetate and lead citrate, and e x a mined with a J e o l 1 0 0 C X TEM. Semithin sections cut o n the s a m e m i c r o t o m e w e r e stained with toluidine b l u e for examination b y light m i c r o s c o p y .
RESULTS
SPATIAL D I S T R I B U T I O N O F HEPATOZOON A N D T I C K S A M O N G L I Z A R D S
A
ll but o n e o f the lizards (n = 1 0 ) caught in the olive grove in Kisra w e r e infected with Hepatozoon kisraen. sp.; o f these, seven w e r e infested with the tick Hyaloma cf. aegyptium ( T a b l e I ) . T h e only lizard found non-infected b y H. kisrae was free from ticks. Ticks r e m o v e d from the H. kisrae-infected lizards hosted either mature sporocysts or premature sporogony stages (Table I I ) , with the exception o f ticks r e m o v e d from the low-infected lizard (No. 2, T a b l e I I ) , w h i c h w e r e not infected. None o f the lizards caught outside the olive grove, either nearby (n = 2 ) , or else
w h e r e (n = 1 5 ) w e r e infected with H. kisrae. T h r e e o f the latter lizards w e r e infected b y the mosquito-trans
mitted Hepatozoon sp. A. Ticks w e r e found o n o n e lizard from site b ( a larva), o n t w o in site c (a nymph and a male, both lizards w e r e also infected with Hepa
tozoon sp. A), and o n two lizards in site d (a male and t w o u n e n g o r g e d females); n o n e w e r e found hosting d e v e l o p i n g stages o f Hepatozoon ( T a b l e I I ) .
DESCRIPTION O F HEPATOZOON KISRAE N . S P .
Hosts: Starred lizard, Agama stellio; the tick Hyalomma cf. aegyptium.
T y p e locality: Olive grove at Kisra (grids 182 E / 1 6 2 N), southeast Samaria, Palestine.
Etymology: Named after the type-host locality.
Stages in the starred lizard
M e r o g o n i c s t a g e s d i v i d i n g b y p o l y e n d o d y o g e n y (Fig. 1A), s e e n in the lungs, w e r e encapsulated (29-39 x 15-22 pm in size, n = 7 ) inside endothelial cells, c o m prised o f either m a c r o m e r o n t s dividing into 4 to 16, 11-16 x 3-4 um merozoites (Fig. 1 A-D), or m i c r o m e - ronts dividing to 16 to 3 2 , 8-11 x 1.5-2 p m merozoites (Fig. 1, A, E - G ) .
In the liver o c c u r r e d the cyst-type meronts or cysto- zoites, with two ("dizoic", 19-24 x 12-17 pm in size, n = 7, Fig. 1H-L), four ( 2 0 - 2 8 x 10-15 pm in size, n - 7, Fig. 2A) and exceptionally eight zoites. T h e s e cysts w e r e lodged within melanomacrophage centers (MMC), evidently inside macrophages. T h e zoite (12-15 x 2-5 um in size, n = 12) c o u p l e s w e r e apparently formed by e n d o d y o g e n y . T h e y w e r e e n c l o s e d in a meront resi
duum (the cystic b o d y ) filled with vacuolar matrix (visible only in histology). S o m e dizoites c o n t a i n e d a blue-staining inclusion, w h i c h might b e a crystalline b o d y (Fig. I I , L). Exceptionally large zoites, 22.2 x 3.8 pm, s h o w e d tapering, d e e p staining, anterior end (not s h o w n ) .
T h e liver also contained a few merogonic stages, mero
g o n i c ( p o l y e n d o d y o g e n i c ) division inside an e n d o thelial cell is s h o w n in Figure 2 B ; m a c r o and micro- m e r o g o n i c stages ( b o t h 2 3 - 2 9 x 1 5 - 1 8 pm in size) c o n t a i n e d up to 16 (Fig. 2 C ) and 32 zoites, respecti
vely. Liver tissue also contained free, single, un-divided meronts (Fig. 2 D ) , reaching a size o f 16-17 x 4-7 pm (n = 2 ) .
Four lizards, two naturally infected, o n e infected by ingestion o f a tick and o n e by infected lizard viscera ( 1 ) , kept for 37 to 167 days, w h e n necropsied, all demonstrated the p r e s e n c e o f cyst-type meronts in the liver. In the two lizards also e x a m i n e d for lung infec-
Infected by No. No. No. With m a t u r e
Lizard m a r k i n g Date H. kisrae ticks p r e s e n t e x a m i n e d infected s p o r o ô c y s t s
1 September 23. 1999 + 0
2 September 23, 1999 i 1M. 2seN 1M, IseN 0
3 September 23, 1999 ++ 1M, IF, 2seN, leL 1M, IF 2 2
(.11 October 15, 1999 0 0
8C May 5, 2000 +++ 3M, IF, 3eL 1M, IF 2 2
9C May 5, 2000 +++ 3M, 4F 1M, 3F 1M, 2F 1M
10C May 5, 2000 ^ + 0
11C May 24, 2000 1M, IF 1M, IF 1M 1
2K August 24, 2000 lOseN 3seN, 1#M 3eN, 1#M 1#M
3K August 24, 2000 + 3seN, 1M 1M, 1#M, 1#F 1M, 1#M, 1#F 1M, 1#M, 1#F
Levels of parasitaemia: ± light (< 0.5 % ) , + low (0.5-3 % ) , ++ moderate (3-9 % ) , +++ high (> 9 % ) .
Table 11. - Analysis of the state of infection of starred lizards and ticks collected in Kisra olive grove (site a); ticks: M-male, F-female, N- nymph, L-larva, e-engorged, se-semiengorged. * Immature ticks were allowed to molt before examination.
PAPERNA I.. KREMER-MECABELL T. & FINKELMAN S.
Fig.1. - Merogonic stages of Hepatozoon kisrae n.sp. from lungs and liver of Agama stellio (x 1,300). A-G, stages in lungs: A. macrome- ronts in division (a) and divided micromeronts (i); B. Macromeronts after division; C. Macromeronts prior division; D. Post-division macro- meront formation; E. Micromeronts prior division; F,G. Post division micromeront formations. H-L: Dizoic cyst-type meronts in the liver (L, merozoites show blue-staining crystalline-like bodies, marked with arrows).
Fig. 2. - Stages of Hepatozoon kisrae n. sp. from Agama stellio (x 1,300). A-D, from the liver: A. Four-zoite cyst-like meront; B. Meront dividing by polyendodyogeny; C. Polyzoic meront (macromeront); D. Undivided meront (arrow) and intraerythrocytic gametocytes (d). E- G: Gametocytes in the blood. E. Oval young (a) and premature with spirale nucleus (c); F. Premature with dense nucleus (b) and mature, encapsulated (d); G. Mature encapsulated; H . Hepatozoon sp. A. in blood of A. stellio from site e.
TlCK TRANSMITTED HEPATOZOON OF AGAMA
PAPERNA L, KREMIER-MECABELL T. & FINKELMAN S.
Lizard m a r k i n g Date infected
Onset o f
parasitaemia Necropsy Liver Lungs Blood
1 natural
Collected September 23, 1999
(before 23.9) after 167 days z2 + Mi & Ma ++ Gam ++
3 natural
Collected September 23, 1999
(before 23.9) after 84 days z2 ++, z4,8 Mi & Ma ++++ Gam ++
2B November 7, 1999
by sporocysts
32 dpi 120 dpi z2 +++, z4, 8, Mi & Ma 16,32 +
? Gam ++
7B March 7, 1999
by lizard viscera
< 37 dpi 37 dpi z2+, z8 ± 7 Gam ++
Table III. - Protocol of necropsies of 4 infected starred lizards: Exoerythrocytic z-zoites (cystozoites), Mi-micromeronts, Ma-macromeronts, Gam-gametocytes in blood. Parasite stages in the tissue: ± scarce, + a few, ++ prevalent, +++ numerous, ++++ veiy numerous.
tion, m a c r o - and micromeront infection coincided with the liver infection ( T a b l e III).
G a m e t o c y t e s at several stages o f differentiation o c c u r red in the erythrocytes. T h e earliest o n e s w e r e oblong, 6-8 x 4-5 pm (n = 4 ) , and s u b s e q u e n t l y oval, 7-9 x 5- 6 pm, with a large central nucleus (Fig. 2 E ) . Interme
diate stages ( 7 - 1 1 x 5-7 pm, n = 4 ) had a nucleus with spirally scattered chromatin (Fig. 2E, F ) . T h e presu
m a b l y fully mature stages w e r e stout, banana-shaped, 12-15 x 2-5.5 p m in size (n = 9 ) , with a centrally l o c a t e d 4 to 5.5 um w i d e multilobed or h o m o g e n e o u s l y p a c k e d nucleus; the cytoplasm was either stained h o m o g e n e o u s l y pale, or had a variable quan
tity o f v a c u o l e s and granules (Fig. 2F, G ) . Fully mature g a m e t o c y t e s w e r e e n c a s e d within a hard capsule. T h e g a m e t o c y t e s w e r e readily differentiated from the sym- patric s p e c i e s o f Hepatozoon found in A. stellio b l o o d (Hepatozoon sp. A, Finkelman & Paperna, unpubli
s h e d ) , the latter being elongate, with inward-bent ends (Fig. 2 H ) .
D e v e l o p m e n t in the tick
Syzygy w a s not detected. Oocysts, 6 7 - 7 4 x 4 7 - 5 4 pm (n = 4 ) in size, with e x p a n d e d vacuolated cytoplasm, and a nucleus with a c o n s p i c u o u s l y large nucleolus, w e r e found in the h e m o c o e l , o n the gut surface o f engorged nymphs (Fig. 3A, B ) . Via segmentation (Fig. 3 0 , sporoblasts formed aggregates o f sporocysts, 4 3 - 5 0 x 24-27 pm (n = 10) in size (Fig. 3 D ) . Early differentiated sporocysts in e n g o r g e d nymphs s h o w e d the already split crystalline b o d i e s ( F i g . 3 E ) . O o c y s t p r o g e n y consisted o f over 100 sporocysts. T h e formed sporo
cysts remained aggregated within the oocyst, forming a ball-like structures ( 2 0 0 - 2 3 0 x 2 3 0 p m in size) in the h e m o c o e l (Fig. 3 F ) visible to the n a k e d e y e as white balls. Mature oocysts with ripe sporocysts w e r e found in u n e n g o r g e d adult male and female ticks and in nymphs, w h i c h failed to c o m p l e t e e n g o r g e m e n t . Indi
vidual sporocysts, e n c a s e d in a hardened wall, varied in size from 3 4 x 24 to 6l x 23 pm, and c o n t a i n e d 16 to 3 5 sporozoites (Fig. 3 G ) .
ULTRASTRIICTURAL OBSERVATIONS ON SPOROCYST DEVELOPMENT
Y o u n g sporocysts w h i c h had split from the sporoblast (Fig. 4 A ) gradually e x h a u s t e d their stored amylopectin granules as well as their lipid v a c u o l e s (Fig. 4 B ) . Small granular aggregates - the anlagen o f crystalloid bodies, e n c l o s e d within rough e n d o p l a s m i c reticulum ( E R ) , grew to large inclusions o f granular matrices (Fig. 4 B , C), w h i c h b e c a m e consolidated into arrays o f crystal
loids (Fig. 4 D , E ) . T h e ER network also incorporated n u m e r o u s tubular mitochondria (Fig. 4 B ) . Small e l e c tron-dense b o d i e s r e m a i n e d in the sporocyst cyto
plasm to late stage of differentiation (Fig. 4A, B , D ) . T h e rough ER, w h i c h a c c o m p a n i e d the forming crys
talloid b o d i e s , disappeared w h e n the crystalloid arrays b e c a m e consolidated (Fig. 4 D , E ) . T h e single nucleus of the formed sporocysts divided. All formed nuclei had a conspicuous, often aggregated nucleolus (Fig. 4 D , E ) . At this stage encapsulation started, the future hard wall consolidated a b o v e the p l a s m a l e m m a , and b e c a m e s u p e r i m p o s e d by a fine veil (Fig. 4 F ) . I n c r e a s e d har
dening o f the sporocyst wall a c c e l e r a t e d its resistence to fixation and impregnation, w h i c h eventually pre
cluded further ultrastructural analysis.
EXPERIMENTAL INFECTIONS From ticks to lizards
Six starred lizards (all adults), from sites other than Kisra, verified to b e non-infected, w e r e fed o n s p o r o - cysts-infected H. cf. aegyptium ticks ( T a b l e IV). Five w e r e fed, e a c h o n a tick r e m o v e d from naturally infected lizard and o n e ( 7 K ) o n a tick experimentally e n g o r g e d o n an infected lizard. T h e latter lizard d e v e l o p e d the s a m e infection s c h e d u l e as the o n e s fed o n naturally infected ticks. All but o n e d e v e l o p e d parasi
temia not later than 32 to 4 0 days post- infection (p.i), in the latter ( 1 C ) , light infection with y o u n g g a m e t o cytes was d e t e c t e d only 9 0 days p.i. In two lizards b l o o d already c o n t a i n e d mature g a m e t o c y t e s b y day
TICK TRANSMITTED HETATOZOON OF AGAMA
Fig. 3. - Developmental stages of Hepatozoon kisrae n. sp. in the tick Hyalomma cf. aegyptium. A-E, toluidine-stained semithin sections;
F,G, unfixed, live: A,B. Oocysts, x 1,156 and x 462; C. Oocyst (o) dividing into sporocysts (s) x 500; D. Sporocysts x 887; E. Sporocysts with divided crystalline bodies x 939; F. Oocyst ball filled with ripe sporocysts x 324; G. Single sporocysts filled with sporozoites x 1,968.
Fig. 4. - Electron microscopic images of sporocysts from infected ticks. A. Newly formed sporocyst filled with amylopectin granules, sho
wing anlagen of crystalloid bodies enclosed in rough ER (pr) mitochondria (m) and electron-dense bodies (d); x 7,150. B. Sporocyst with ER-aligned inclusion of pre-crystalloid granular bodies (r), showing also mitochondria (m) and lipid vacuoles (L); x 6,400. C. Sector of divided sporocysts showing a nucleus (n) with scattered nucleolus (ns), and ER (er) aligning the pre-crystalloid granular body (r): x 10,344.
D,E. Sporocysts with several nuclei (n), arrayed crystalloid bodies (r), amylopectin granules (a) and electron-dense bodies (d); x 6000 and X 6, 460. F. Walling process of a sporocyst, the forming wall is invested by a veil (arrowhead); In addition to the formed crystalloid body (r) a crystalloid anlage (pr) enclosed in ER still occurs; a, amylopectin granules; x 15,300.
PAPERNA I., KREMER-MECABELL T. & FINKELMAN S.
TICK TRANSMITTED HEPATOZOON OF AGAMA
Date fed Dpi to first Dpi to m a t u r e
Lizard m a r k i n g Origin Date o n s p o r o c y s t s s e e n infection gametocytes
2B site d October 15, 1999 November 7, 1999 32 60
IC site c May 5, 2000 May 15, 2000 93 ~
IK site b August 24, 2000 October 17, 2000 40
4K site b August 24, 2000 October 26, 2000 40
5K site e August 24, 2000 September 3, 2000 37
7K site d August 24, 2000 September 3. 2000 < 37 37 (died)
37 p.i., in further t w o by day 4 0 p.i. and in o n e b y day 60 p.i.
T h e t w o g e c k o e s fed simultaneously with I K and 4K starred lizards on sporocyst-infected tick viscera, failed to d e v e l o p infection b y day 4 0 p.i.
From starred lizard to starred lizard
T h r e e lizards ( t w o juveniles and o n e adult from out
side Samaria) w e r e fed o n b l o o d and liver o b t a i n e d from a euthanized infected lizard (lizard 1, T a b l e I I ) . Parasitaemia, including o f mature e n c a p s u l a t e d g a m e tocytes was detected b y day 37 in the adult and 47 days p.i. in b o t h juvenile lizards.
From lizards to tick
T w o H. k i s r a e - i n f e c t e d lizards, and three n o n infected o n e s w e r e e x p o s e d to H. cf. aegyptium larvae, engor
g e d larvae w e r e o b t a i n e d after 11-13 days p o s t - e x p o sure, s o m e o f the larvae p r o c e e d e d without d e s c e n ding to the n y m p h stage and d r o p p e d off as e n g o r g e d nymphs 3 0 to 3 8 days later. N o n e o f these, engorged larvae o r hungry nymphs, r e c o v e r e d up to 4 0 days post-attachment w e r e found infected. Infection was, h o w e v e r , found in s p o n t a n e o u s l y d e t a c h e d s e m i - e n g o r g e d n y m p h s (n = 6) w h o failed to molt, exa
m i n e d 41 days post initial attachment. Infection was already c o m p r i s e d o f mature sporocysts with sporo- zoites. Adult ticks developing from molting e n g o r g e d n y m p h s w e r e f o u n d i n f e c t e d b y n o n - s p o r u l a t e d oocysts, as well as oocysts containing non-differentiated and mature sporocysts. T h e s e ticks w e r e 41 to 77 days after initial e x p o s u r e .
All 10 dissected adult ticks and three engorged nymphs r e c o v e r e d after a feeding s c h e d u l e o n three Hepato
zoon sp. A.-infected lizards e x a m i n e d either 42 to 77 days (adults) or 2 0 to 42 days ( e n g o r g e d n y m p h s ) after initial attachment w e r e negative. O n e engorged nymph e x a m i n e d 22 days post-attachment c o n t a i n e d Hepato
zoon sp. A. g a m e t o c y t e s . Transovarian transmission
Infection w a s not traced in any o f the p r o g e n y grown from ovigerous female r e m o v e d from infected lizard from Kisra. E x a m i n e d ticks w e r e n y m p h s ( 3 ) and adults ( 1 0 ) e n g o r g e d after f e e d i n g o n three n o n - infected lizards.
DISCUSSION
A gama stellio, the starred lizard is abundant and widely distributed in the Near East and has
become
a peridomestic inhabitant (Haas, 1951).O n the otherhand the spatial distribution o f both ticks and H. kisrae infection in starred lizards appears to b e very patchy, the present locality was restricted to a stone fence-enclosed area o f less than 1,000 m2. Desser
& Yekutiel ( 1 9 8 6 - 1 9 8 7 ) reported Hepatozoon infection b y stout g a m e t o c y t e s , a p p a r e n t l y c o n s p e c i f i c with H. kisrae, in starred lizards from three localities: two in the s a m e arid geographical subregion (in south-east Samaria), and o n e in the forested Mediterranean z o n e in west Jerusalem, with ~ 8 0 0 mm annual rainfall. Hya- lomma cf. aegyptium w e r e n e v e r b e e n found o n the previously investigated starred lizards (Ostrovska &
Paperna, 1987; Bristowetzki & Paperna, 1 9 9 0 ) . As was found in our study, not all p a t c h e s o f tick infection necessarily generate active H. kisrae transmission. T h e s p e c i e s o f Hepatozoon co-habiting starred lizards are readily distinguishable, and appears to demonstrate a strict specificity to their respective vector hosts, H. kis
rae, with the stout gametocytes, to H. cf. aegyptium and Hepatozoon sp. A, with the elongate gametocytes, to m o s q u i t o e s (Culex pipiens, Finkelman & Paperna, unpublished). Transmission by mosquitoes could favor a m o r e continuous pattern o f distribution for Hepato
zoon sp. A. T h e spatial distribution o f this apparently m o r e a b u n d a n t s p e c i e s , h o w e v e r , is a l s o p a t c h y , though over seemingly a wider range o f habitats (Fin
kelman & Paperna, unpublished).
H. kisrae appears to have narrow host specificity, p o s sibly only A. stellio, infection failed to established itself in g e c k o e s (P. hasselquistii) fed o n infected ticks.
T h e vector tick's identity remains inconclusive. T h e ticks found o n the lizards, by all morphological criteria outlined by Hoogstraal ( 1 9 5 6 ) , are indistinguishable from H. aegyptium, w h i c h has b e e n found feeding pre
dominantly o n land tortoises (Testudo graeca) and vectors Hemolivia mauritanica (Michel, 1 9 7 3 ; Landau
& Paperna, 1 9 9 7 ) . Although Hoogstraal ( 1 9 5 6 ) notes that rarely the tick w a s found feeding o n A. stellio, in our e x p e r i m e n t s ( u n p u b l i s h e d ) , larvae, nymphs and Table IV. - Experimental infection of starred lizards by feeding on H. kisrae sporocysts from ticks.
P A P E R N A I., K R E M E R - M E C A B E L L T . & F I N K E L M A N S.
adults reared from the ticks recovered from the lizards, refused to attach to tortoises.
Ball et al. ( 1 9 6 9 ) found sporozoite stages in the tick Amblyoma dissimile as well as in m o s q u i t o e s (Culex tarsalis, Aedes togoito), w h e n fed o n s a m e Hepatozoon fusifex-'mkcted Boa constrictor. T h e parasites, w h i c h
d e v e l o p e d in the tick, as authors also admit, h o w e v e r , do not necessarily have to b e conspecific with the o n e s found in the m o s q u i t o e s . T h e s e m o s q u i t o e s failed to acquire infection w h e n fed on s n a k e s infected via A. dissimile.
Apparently, all true m e m b e r s o f the genus Hepatozoon, as well as s p e c i e s o f Hemolivia have a d i c h o t o m o u s course o f merogony, o n e o f active large-progeny mero- g o n i e s to sustain the s u b s e q u e n t g a m o g o n o u s g e n e ration in the b l o o d and the other, yielding persistant c y s t - l i k e s t a g e s . T h e s e c y s t - e n c l o s e d s t a g e s w e r e termed cystozoites (Landau et al., 1 9 7 2 ) . T h e y w e r e transmitted w h e n ingested as prey by a subsequent ver
tebrate host (Landau et al, 1972; Smith & Desser, 1998).
In our experiments, active m e r o g o n i c stages could not b e separated from the cyst-like stages in the viscera used to infect lizards via feeding.
In H. kisrae, active m e r o g o n i e s d e v e l o p e d in e n d o t h e lial cells in the lungs and to a lesser extent in the liver.
Cystozoites o c c u r only in the liver, mostly in m a c r o p h a g e s in MMC. In A. stellio experimentally infected by Hemolivia mariae, a few cystozoites also developed in the lungs, in formed MMC (Smallridge & Paperna, unpublished).
T h e large-progeny m e r o g o n i e s are formed by polyen- d o d y o g e n o u s m e r o g o n y , and generations o f 4 to 16 large meronts are followed by o n e or m o r e eight to 32 progeny-generations o f micromeronts, which e s c a p e to the b l o o d to d e v e l o p into g am e t o cy t e s. T h e dizoic cystozoites have b e e n s e e n to form by e n d o d y o g e n y (Landau et al, 1 9 7 2 ) , as also s e e n in this study: cystic stage-four and eight zoites are g e n e r a t e d b y s u c c e s sive endoclyogenies.
D e v e l o p m e n t o f the p r e s e n t l y d e s c r i b e d s p e c i e s , although taking place in ticks, o c c u r s similarly to that seen in moquitoes, i.e. within the oocyst located in the tissue lining the h a e m o c o e l (Bashtar etal, 1984; Lowi- chik etal, 1993; Smith & Desser, 1 9 9 7 ) . In Hemolivia developing in ticks the sporocysts released (as sporo- k i n e t e s ) from the oocyst remain located inside parasi- tophorous vacuoles within the tick gut cells (Smallridge
& Paperna, 2 0 0 0 b ) .
In ultrastnicturally e x a m i n e d H. kisrae sporocysts, the crystalloid material initially a s s e m b l e d within p o c k e t s o f E R , by the s a m e route as s e e n in s p e c i e s d e v e l o ping in m o s q u i t o e s (H. domerguei, Vivier etal, 1972;
H. aegyptii, Bashtar et al, 1 9 8 4 ) , and similarly led to the formation o f large crystalloid arrays w h i c h are split b e t w e e n t h e u l t i m a t e l y f o r m e d s p o r o z o i t e s . Although the developmental process in Hemolivia also
involves formation and split o f crystalloid substance b e t w e e n the offspring, it s e e m s to p r o c e e d differently.
T h e extensive E R , w h i c h a c c o m p a n i e s the forming crystalloid b o d i e s in Hepatozoon, is lacking in Hemo
livia (or disappears at an earlier stage o f differentia
tion). In Hemolivia the crystalloid substance is first split b e t w e e n sporokinetes (Smallridge & Paperna, 2 0 0 0 a ) , and only then b e t w e e n the sporozoites (Paperna &
Smallridge, 2 0 0 0 b ) .
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Regu le 27 septembre 2001 Accepte le 12 novembre 2001
