Zeitschrift far
Parasitenkunde
Z. Parasitenkd. 58, 191-194(1979)
Parasitology Research
9 by Springer-Verlag 1979
Electron Microscopic Detection of Initial and Some Subsequent Developmental Stages of Babesia bigemina in Salivary Glands of Ticks Giinter W e b e r * and Karl Friedhoff Protozoologische Abteilung, Institut ffir Parasitologie, Tier/irztliche Hochschule Hannover, Btinteweg 17, D-3000 Hannover 71
Light microscopic w o r k on Babesia in salivary glands of ticks (Christophers, 1907; Dennis, 1932; C a p r u n , 1957; Li, 1958; Riek, 1964) indicates that development o f the parasites in this tissue starts f r o m kinetes I which have invaded the glandular cells. These workers agree that the kinetes r o u n d up, increase in size, and u n d e r g o nuclear divisions. The cytoplasmic mass becomes progressively fragmented until sporozoitesl are formed. Ultrastructures of the latter have been described in B. ovis (Friedhoff et al., 1972) and in B. boris (Potgieter and Els, 1976). In our experiments nymphs, which developed f r o m transovarially infected larvae of Boophilus microplus, were dissected twelve days after infestation on a calf and submersed in 2% p a r a f o r m a l d e h y d e (PFA) in 0.1 M cacodylate buffer, p H 7.4. The salivary glands were removed, fixed for ten hours ( 2 0 ~ and 4 ~ in a mixture o f 2% P F A and 2% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in OsO4, and embedded in Epon. Parasites were encountered in cytoplasmic infoldings of cells free of secretory granules (Fig. 1). There were n u m e r o u s kinetes which showed the same structures (pellicular complex, apicalschirm, and others) as had been observed previously in kinetes of this species in tick h e m o l y m p h (Weber, 1977) and ovary (Friedhoff, Scholtyseck, 1969) (Figs. 2 and 3). The developmental phase observed includes the following steps: (1) r o u n d i n g up and *
To whom offprint requests should be sent
1 This term is used on the basis of our own tentative interpretation and is not necessarily the same as used in the original paper
Abbreviations m m1 m2 m3 mi n
= = = = = =
micronemes outer ] middle / pellicular membrane inner parasite mitochondrion parasite nucleus
p r S wn x
= = = = =
parasite rhoptry spheroid body host cell nucleus cytoplasmic invagination
0044-3255/79/0058/0191/$01.00
192
G. Weber and K. Friedhoff
Electron Microscopic Detection of Initial
193
e n l a r g e m e n t o f the kinete a c c o m p a n i e d by r e d u c t i o n o f the t r i p l e - m e m b r a n e d pellicular c o m p l e x t o a s i n g l e - m e m b r a n e d pellicle, a n d other processes o f d i f f e r e n t i a t i o n (Figs. 1, 4 a n d 5), (2) n u c l e a r division a n d p a r t i t i o n o f the p a r a s i t e in m u l t i - n u c l e a t e d b o d i e s (Fig. 6), a n d (3) f o r m a t i o n o f singlen u c l e a t e d s p o r o z o i t e s (Fig. 7). S p o r o z o i t e s were e n c o u n t e r e d in great n u m b e r s either densely p a c k e d in s o m e ceils (Fig. 7) or loosely d i s p e r s e d in others (Fig. 1). M o r p h o l o g i c characteristics o f these stages are: l o n g i s h - p i r i f o r m shape, t r i p l e - m e m b r a n e d pellicle, one nucleus, apical m i c r o n e m e s , r h o p t r i e s in u n c e r t a i n n u m b e r s , one or m o r e ' s p h e r o i d b o d i e s ' (Figs, 7, 8 a n d 9). The latter were r a t h e r electron-dense, s p o n g i f o r m in a p p e a r a n c e , a n d b o u n d e d by a single i n c o n s p i c u o u s m e m b r a n e . Typically, the s p h e r o i d b o d i e s s h o w e d a deep inv a g i n a t i o n o f the c y t o p l a s m (Figs. 7 a n d 9). T h e y v a r i e d c o n s i d e r a b l y in size a n d shape. O u r o b s e r v a t i o n s c o n f i r m the light m i c r o s c o p i c w o r k cited above. E l e c t r o n m i c r o s c o p i c studies on details o f structures a n d d e v e l o p m e n t o f B. bigemina in tick salivary g l a n d s are in progress.
Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft.
References Caprun, A.A. : Untersuchungsergebnisse fiber die Entwicklung yon Piroplasma caballi in Zecken der Gattung Dermacentor (in Russian). Trudy Vsesojuz. Inst. Eksp. Vet. 21, 221-240 (1957) Christophers, S.R. : Piroplasma canis and its life cycle in the tick. Scientific memoirs by officers of the medical and sanitary departments of the government of India (new series) No. 29. Office of the superintendent of government printing, Calcutta: 1907
Fig. I. Part of a salivary gland alveokls of a Boophitus microptus nymph infected with Babesia bigemina. Enlarged, rounded kinetes (arrows) and fission bodies in advanced stages of sporozoite formation (double-arrows). x 850 Fig. 2. Rounded kinetes between infoldings of the host cell cytoplasm, x 8,500 Fig. 3. Anterior end of a kinete which has still retained the typical aspect of a hemolymphal kinete, x 20,000 Fig. 4. In this parasite the apical rhoptries are still visible (arrows), but the pellicular complex has already been reduced to a single membrane (plasmalemma). x 12,000 Fig. 5. Section through a rounded kinete parts of whose pellicle stilt show an inner layer (double-arrows); the other part is single-membraned (arrows). • 12,000 Fig. 6. Multi-nucleated body (top). x 17,000 Fig. 7. Densely packed, differentiated sporozoites, x 18,000 Fig. 8. Part of the triple-membraned pellicle of a sporozoite, x 100,000 Fig. 9. Diagrammatic representation of a sporozoite of Babesia bigemina in salivary glands of nymphal Boophilus rnicroplus ticks
194
G. Weber and K. Friedhoff
Dennis, E.W.: The life-cycle of Babesia bigemina (Smith and Kilbourne) of Texas cattle-fever in the tick Margaropus annulatus (Say). Univ. Calif. Publ. Zool. 36, 263-298 (1932) Friedhoff, K.T., Scholtyseck, E. : Feinstrukturen der Merozoiten yon Babesia bigemina im Ovar yon Boophilus microplus und Boophilus decoloratus. Z. Parasitenkd. 32, 266 283 (1969) Friedhoff, K.T., Scholtyseck, E., Weber, G. : Die Feinstruktur der differenzierten Merozoiten yon Babesia ovis in den Speicheldrt~sen weiblicher Zecken. Z. Parasitenkd. 38, 132-140 (1972) Li, P.N.: Die Wechselbeziehungen zwischen dem Blutsaugen infizierter Zecken der Art Rhipicephalus bursa und der Entwicklung des Erregers der Schafbabesiellose, Babesiella ovis, in ihnen (in Russian). Trudy Ukrain. Naut. Inst. Eksp. Vet. 24, 271 282 (1958) Potgieter, F.T., Els, H.J.: Light and electron microscopic observations on the development of smaiI merozoites of Babesia boris in Boophitus microptus larvae. Onderstepoort J. Vet. Res. 43, 123-128 (1976) Rick, R.F.: The life-cycle of Babesia bigemina (Smith and Kilbourne, 1893) in the tick vector Boophilus microplus (Canestrini). Aust. J. Agric. Res. 15, 802-821 (1964) Weber, G. : Ultra-morphology and cytochemistry of developmental stages of Babesia bigemina and B. ovis (Apicomplexa: Piroplasmea) in tick hemolymph. Proceedings of the Vth International Congress of Protozoology, New York (1977)
Received September 30, 1978
Parasitenkunde
Z. Parasitenkd. 58, 191-194(1979)
Parasitology Research
9 by Springer-Verlag 1979
Electron Microscopic Detection of Initial and Some Subsequent Developmental Stages of Babesia bigemina in Salivary Glands of Ticks Giinter W e b e r * and Karl Friedhoff Protozoologische Abteilung, Institut ffir Parasitologie, Tier/irztliche Hochschule Hannover, Btinteweg 17, D-3000 Hannover 71
Light microscopic w o r k on Babesia in salivary glands of ticks (Christophers, 1907; Dennis, 1932; C a p r u n , 1957; Li, 1958; Riek, 1964) indicates that development o f the parasites in this tissue starts f r o m kinetes I which have invaded the glandular cells. These workers agree that the kinetes r o u n d up, increase in size, and u n d e r g o nuclear divisions. The cytoplasmic mass becomes progressively fragmented until sporozoitesl are formed. Ultrastructures of the latter have been described in B. ovis (Friedhoff et al., 1972) and in B. boris (Potgieter and Els, 1976). In our experiments nymphs, which developed f r o m transovarially infected larvae of Boophilus microplus, were dissected twelve days after infestation on a calf and submersed in 2% p a r a f o r m a l d e h y d e (PFA) in 0.1 M cacodylate buffer, p H 7.4. The salivary glands were removed, fixed for ten hours ( 2 0 ~ and 4 ~ in a mixture o f 2% P F A and 2% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in OsO4, and embedded in Epon. Parasites were encountered in cytoplasmic infoldings of cells free of secretory granules (Fig. 1). There were n u m e r o u s kinetes which showed the same structures (pellicular complex, apicalschirm, and others) as had been observed previously in kinetes of this species in tick h e m o l y m p h (Weber, 1977) and ovary (Friedhoff, Scholtyseck, 1969) (Figs. 2 and 3). The developmental phase observed includes the following steps: (1) r o u n d i n g up and *
To whom offprint requests should be sent
1 This term is used on the basis of our own tentative interpretation and is not necessarily the same as used in the original paper
Abbreviations m m1 m2 m3 mi n
= = = = = =
micronemes outer ] middle / pellicular membrane inner parasite mitochondrion parasite nucleus
p r S wn x
= = = = =
parasite rhoptry spheroid body host cell nucleus cytoplasmic invagination
0044-3255/79/0058/0191/$01.00
192
G. Weber and K. Friedhoff
Electron Microscopic Detection of Initial
193
e n l a r g e m e n t o f the kinete a c c o m p a n i e d by r e d u c t i o n o f the t r i p l e - m e m b r a n e d pellicular c o m p l e x t o a s i n g l e - m e m b r a n e d pellicle, a n d other processes o f d i f f e r e n t i a t i o n (Figs. 1, 4 a n d 5), (2) n u c l e a r division a n d p a r t i t i o n o f the p a r a s i t e in m u l t i - n u c l e a t e d b o d i e s (Fig. 6), a n d (3) f o r m a t i o n o f singlen u c l e a t e d s p o r o z o i t e s (Fig. 7). S p o r o z o i t e s were e n c o u n t e r e d in great n u m b e r s either densely p a c k e d in s o m e ceils (Fig. 7) or loosely d i s p e r s e d in others (Fig. 1). M o r p h o l o g i c characteristics o f these stages are: l o n g i s h - p i r i f o r m shape, t r i p l e - m e m b r a n e d pellicle, one nucleus, apical m i c r o n e m e s , r h o p t r i e s in u n c e r t a i n n u m b e r s , one or m o r e ' s p h e r o i d b o d i e s ' (Figs, 7, 8 a n d 9). The latter were r a t h e r electron-dense, s p o n g i f o r m in a p p e a r a n c e , a n d b o u n d e d by a single i n c o n s p i c u o u s m e m b r a n e . Typically, the s p h e r o i d b o d i e s s h o w e d a deep inv a g i n a t i o n o f the c y t o p l a s m (Figs. 7 a n d 9). T h e y v a r i e d c o n s i d e r a b l y in size a n d shape. O u r o b s e r v a t i o n s c o n f i r m the light m i c r o s c o p i c w o r k cited above. E l e c t r o n m i c r o s c o p i c studies on details o f structures a n d d e v e l o p m e n t o f B. bigemina in tick salivary g l a n d s are in progress.
Acknowledgement. This work was supported by the Deutsche Forschungsgemeinschaft.
References Caprun, A.A. : Untersuchungsergebnisse fiber die Entwicklung yon Piroplasma caballi in Zecken der Gattung Dermacentor (in Russian). Trudy Vsesojuz. Inst. Eksp. Vet. 21, 221-240 (1957) Christophers, S.R. : Piroplasma canis and its life cycle in the tick. Scientific memoirs by officers of the medical and sanitary departments of the government of India (new series) No. 29. Office of the superintendent of government printing, Calcutta: 1907
Fig. I. Part of a salivary gland alveokls of a Boophitus microptus nymph infected with Babesia bigemina. Enlarged, rounded kinetes (arrows) and fission bodies in advanced stages of sporozoite formation (double-arrows). x 850 Fig. 2. Rounded kinetes between infoldings of the host cell cytoplasm, x 8,500 Fig. 3. Anterior end of a kinete which has still retained the typical aspect of a hemolymphal kinete, x 20,000 Fig. 4. In this parasite the apical rhoptries are still visible (arrows), but the pellicular complex has already been reduced to a single membrane (plasmalemma). x 12,000 Fig. 5. Section through a rounded kinete parts of whose pellicle stilt show an inner layer (double-arrows); the other part is single-membraned (arrows). • 12,000 Fig. 6. Multi-nucleated body (top). x 17,000 Fig. 7. Densely packed, differentiated sporozoites, x 18,000 Fig. 8. Part of the triple-membraned pellicle of a sporozoite, x 100,000 Fig. 9. Diagrammatic representation of a sporozoite of Babesia bigemina in salivary glands of nymphal Boophilus rnicroplus ticks
194
G. Weber and K. Friedhoff
Dennis, E.W.: The life-cycle of Babesia bigemina (Smith and Kilbourne) of Texas cattle-fever in the tick Margaropus annulatus (Say). Univ. Calif. Publ. Zool. 36, 263-298 (1932) Friedhoff, K.T., Scholtyseck, E. : Feinstrukturen der Merozoiten yon Babesia bigemina im Ovar yon Boophilus microplus und Boophilus decoloratus. Z. Parasitenkd. 32, 266 283 (1969) Friedhoff, K.T., Scholtyseck, E., Weber, G. : Die Feinstruktur der differenzierten Merozoiten yon Babesia ovis in den Speicheldrt~sen weiblicher Zecken. Z. Parasitenkd. 38, 132-140 (1972) Li, P.N.: Die Wechselbeziehungen zwischen dem Blutsaugen infizierter Zecken der Art Rhipicephalus bursa und der Entwicklung des Erregers der Schafbabesiellose, Babesiella ovis, in ihnen (in Russian). Trudy Ukrain. Naut. Inst. Eksp. Vet. 24, 271 282 (1958) Potgieter, F.T., Els, H.J.: Light and electron microscopic observations on the development of smaiI merozoites of Babesia boris in Boophitus microptus larvae. Onderstepoort J. Vet. Res. 43, 123-128 (1976) Rick, R.F.: The life-cycle of Babesia bigemina (Smith and Kilbourne, 1893) in the tick vector Boophilus microplus (Canestrini). Aust. J. Agric. Res. 15, 802-821 (1964) Weber, G. : Ultra-morphology and cytochemistry of developmental stages of Babesia bigemina and B. ovis (Apicomplexa: Piroplasmea) in tick hemolymph. Proceedings of the Vth International Congress of Protozoology, New York (1977)
Received September 30, 1978
