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The integrity of host lymphocyte plasmalemma during its invasion by Theileria annulata sporozoites as demonstrated by ruthenium red staining
Veterinary Parasitology, 17 (1984/85) 279--286 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
279
THE INTEGRITY OF HOST LYMPHOCYTE PLASMALEMMA DURING ITS I N V A S I O N BY T H E I L E R I A A N N U L A T A S P O R O Z O I T E S AS D E M O N S T R A T E D BY R U T H E N I U M R E D S T A I N I N G
W.G.Z.O. JURA Veterinary Research Department, K.A.R.I., Muguga, P.O. Box 32, Kikuyu (Kenya) (Accepted 26 October 1984)
ABSTRACT Jura, W.G.Z.O., 1985. The integrity of host lymphocyte plasmalemma during its invasion by Theileria annulata sporozoites as demonstrated by ruthenium red staining. Vet. Parasitol., 17: 279--286. The integrity of host lymphocyte plasma membrane during its invasion by Theileria annulata sporozoites iwas examined in an in vitro system using ruthenium red staining. It was demonstrated that during interiorisation of the sporozoites, the dye was completely excluded from the intracellular milieu, indicating that the continuity of the host-ceU plasmalemma was not disrupted throughout the process of penetration, and confirmed a previous observation that the fragmentation of the host lymphocyte membrane only occurs later, after the parasite is intracellular. INTRODUCTION Previous s t u d i e s have s h o w n t h a t Theileria annulata (Jura, 1981; J u r a et al., 1 9 8 3 ) and T. parva ( F a w c e t t et al., 1 9 8 2 ) s p o r o z o i t e s interiorise b y d e e p e n i n g an invagination o f the target b o v i n e - l y m p h o c y t e m e m b r a n e . T h e host-cell m e m b r a n e investing the interiorised s p o r o z o i t e f r a g m e n t s within 30 min o f e n t r y so t h a t the parasite remains in direct c o m m u n i c a t i o n with the c y t o s o l o f the h o s t l y m p h o c y t e ( J u r a et al., 1983). W h e t h e r some damage o c c u r s t o the h o s t l y m p h o c y t e p l a s m a l e m m a during the e n t r y o f sporozoites has n o t b e e n conclusively d e t e r m i n e d . In this s t u d y , the integrity o f the h o s t l y m p h o c y t e m e m b r a n e during the interiorisation o f T. annulata s p o r o z o i t e s was e x a m i n e d using r u t h e n i u m red staining. T h e m e c h a n i s m o f a c t i o n o f r u t h e n i u m red (Mangin, 1 9 8 3 ) , [RU302(NH3)14] C16"4H20 ( F l e t c h e r et al., 1961) was studied and discussed b y L u f t (1971). T h e d y e binds to extracellular acid m u c o p o l y s a c c h a r i d e s and related substances with o s m i u m t e t r o x i d e (OsO4) providing general f i x a t i o n and low c o n t r a s t so t h a t brownish-black deposits localise at sites w h e r e t h e r u t h e n i u m red--OsO4-coupled r e a c t i o n occurs. T h e first e v e n t is t h e linking o f t h e
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© 1985 Elsevier Science Publishers B.V.
280 ruthenium red to the acidic polysaccharides of tissues, whereby the specificity of the reaction is expressed. The ruthenium red is then oxidised by OsO4 in the mixture of ruthenium brown which in turn oxidises the polysaccharide to which it is bound. The overall reaction is an oxidation of the polysaccharide with an equivalent reduction of OsO4 to lower insoluble oxidation products which are localised extracellularly. The exclusion of ruthenium red--OsO4 reaction products delineates cell membranes and permits tracing tortuous plasmalemmal invaginations so that no intracellular staining is possible unless the continuity of the cell membrane is disrupted. MATERIALS AND METHODS
Preparation o f T. annulata sporozoite and peripheral blood lymphocyte (PBL ) suspensions and establishment o f cultures Details of the methods, modified after Brown (1979), by which PBL were separated from bovine venous blood, and by which sporozoites of T. annulata were prepared from infected ticks, have been described elsewhere (Jura et al., 1983). In brief, 3-day.fed Hyalomma anatolicum anatolicum ticks infected with T. annulata were surface-sterilised and ground in 3.5% bovine plasma albumin (Armour fraction V, Sigma Chemical Company, St. Louis, Missouri) in Eagle's minimum essential medium (MEM) (Gibco--Europe) to provide ground up tick supernate (GUTS) of a concentration equivalent to 2 ticks m1-1 and filtered to 8/1 (GUTS filtrate). PBL suspension was prepared from b u f f y coat obtained from defibrinated blood and layered on to a Ficoll/sodium diatrizoate gradient (Ficoll-plaque, Pharmacia Fine Chemicals, Uppsala, Sweden). The cell concentration was adjusted to 8 × 106 cells ml -~ of growth medium-RPMI 1640 with 20% foetal calf serum (Gibco--Europe), containing 100 i.u. m1-1 benzylpenicillin, 100 tlg ml -~ streptomycin sulphate (Glaxo Laboratories Limited, Greenford, England) and 2 mM ml -~ L-glutamine (Gibco-Europe). Cultures were established in triplicate by mixing 0.25 ml GUTS filtrate with 0.25 ml PBL suspension. Plates containing cultures were placed in a humidified box, gassed with 5% CO2/95% air and incubated at 37°C.
Preparation o f stock solution o f ruthenium red Commercial ruthenium red (50 mg) (John Mathey and Company Inc., 608 Fifth Avenue, New York, U.S.A.) was weighed, crushed in a mortar and a few drops of deionised, distilled water were added while continuing the crushing. A total of 5 ml distilled water was added to make 10 mg m1-1 final concentration, and the suspension transferred to a 15-ml centrifuge tube and heated in a water bath at 60°C with frequent agitation for 5 min then centrifuged at 1500 g for 10 min at 20°C (3000 r. min -1, MSE Chilspin). The supernate was stored at 4°C as a stock solution.
281
Procedure for ruthenium red staining The ruthenium red technique was applied to samples obtained at 15, 30 and 60 min of incubation to the technique described by Luft (1971). Suspensions of cells were centrifuged at 275 g for 10 min at 20°C {1450 r. min -~, MSE Minor), and pellets fixed for 1 h at 4°C in a freshly-prepared mixture of 0.5 ml 0.2 M cacodylate-buffered glutaraldehyde, 0.5 ml 0.2 M cacodylate buffer, pH 7.4 and 0.5 ml stock solution of ruthenium red. The pellets were then rinsed in 3 changes of 0.2 M cacodylate buffer over a period of 10 min and fixed again at 20°C for 3 h in freshly-prepared mixture of 0.5 ml 5% OsO4 in distilled water, 0.5 ml 0.2 M cacodylate buffer and 0.5 ml stock solution of ruthenium red. They were then washed in 3 changes of 0.2 M
Fig. 1. A non-infected ~bovine lymphocyte fixed and post fixed in glutaraldehyde-ruthenium red and OsO~--ruthenium red mixtures, respectively, and processed to araldite by routine electron microscopic techniques. Complete localisation of reaction products of ruthenium red, with distinct delineation of lymphocyte plasmalemma is demonstrated by increased electron density ( ~ ' ) of the membrane. Magnification, X 20 87
282 c a c o d y l a t e buffer, d e h y d r a t e d in graded series o f e t h a n o l and p r o c e s s e d to araldite. Sections, c u t w i t h L K B glass knives on a C a m b r i d g e - H u x l e y Ultram i c r o t o m e , w e r e m o u n t e d on c o p p e r grids, stained w i t h u r a n y l a c e t a t e (Watson, 1 9 5 8 ) and lead citrate ( R e y n o l d s , 1 9 6 3 ) and e x a m i n e d on a Philips 400 electron microscope. RESULTS C o m p l e t e e x t r a c e l l u l a r localisation o f r e a c t i o n p r o d u c t s o f r u t h e n i u m red, w i t h distinct d e l i n e a t i o n of a n o n - i n f e c t e d l y m p h o c y t e p l a s m a l e m m a was d e m o n s t r a t e d b y increased e l e c t r o n d e n s i t y o f t h e m e m b r a n e (Fig. 1). O b s e r v a t i o n s d u r i n g t h e various stages o f i n t e r i o r i s a t i o n o f T. a n n u l a t a
Fig. 2. A T. annulata sporozoite in the process of interiorisation and stained with ruthenium red is illustrated. The target lymphocyte plasmalemma is deeply invaginated so that its ends (.~,,m) are close to fusing around the invading parasite. The lymphocyte membrane circumjacent to the sporozoite ( ~ ) remains intact throughout the process as demonstrated by lack of intracellular ruthenium red staining. A portion of host nucleus ( HN ) is shown. Magnification, x 128 590.
283 sporozoites (Figs. 2 and 3) showed that the cell membrane circumjacent to the invading parasite remained intact so that no intraceUular ruthenium red--OsO4 reaction products were observed. In cases where the sporozoites had been completely interiorised, and the invaginated host membrane fused and sealed before staining (Fig. 4), so t h a t there was no communication with extracellular milieu, no ruthenium red was demonstrated in the host plasma membrane investing the parasite. DISCUSSION In this study, it has been demonstrated by ruthenium red staining that when T. annulata sporozoites invade and enter bovine lymphocytes, the dye is completely excluded from the intracellular milieu, indicating that the continuity of the plasmalemma of the host cell is not disrupted, and remains
Fig. 3. A T. annulata sporozoite processed with ruthenium red-fixative mixtures in the course of interiorisation is almost completely intracellular. The ends ( ~ ) of the invaginating target l y m p h o c y t e membrane are touching, but not yet fused. The investing host cell membrane as well as parasite pellicle are delineated by electron-dense ruthenium red--OsO 4 reaction products ( ~ = : ) . No reaction products are intracellular, coni~nTning that T. annulata sporozoites do not disrupt target cell membrane during the entry process. Magnification, × 115 500.
284 intact throughout the process of interiorisation. However, a short while after the parasite is intracellular, the circumjacent host membrane has been shown to break up and disappear (Jura et al., 1983). Similarly, certain members of the phylum Apicomplexa such as Selenidium hollandei (Metals ~nd Schrevel, 1974), Eimeria magna (Jensen and Hammond, 1975; Jensen and Edgar, 1976) and mammalian malaria parasites, Plasmodium berghei and P. knowlesi (Danforth et al., 1980) have also been shown to achieve interiorisation by active invasion and invagination of the host-cell membrane without disrupting its continuity. H o w the parasites accomplish the invagination of their host membranes is no t clear, but several suggestions have been made. Russel (1983) suggested that Eimeria tenella and E. acervulina enter the parasitophorous vacuole by "capping" the host/parasite junction down their body, so locomoting into the host cell. He believed the mechanism required no additional modifications to either cell, was facilitated by the specificity of
Fig. 4. An electron micrograph s h o w i n g intracellular and attached T. annulata sporozoites processed with r u t h e n i u m red--fixative mixture. The intracellulax parasite interiorised prior to r u t h e n i u m red treatment hence no reaction products axe demontsrated either on its pellicle ( P ) or on the circumjaeent host membrane. The attached sporozoite and the l y m p h o c y t e membranes are densely covered with r u t h e n i u m red--OsO 4 reaction products (,qJ====). Magnification, x 43 250.
285 m e m b r a n e r e c e p t o r s , and the parasite's c o n t r a c t i l e s y s t e m was the p r i m e " m o v e r " . J u r a ( 1 9 8 3 ) o b s e r v e d t h a t following a firm r e c e p t o r - d e p e n d e n t a t t a c h m e n t o f T. annulata s p o r o z o i t e s t o t a r g e t l y m p h o c y t e m e m b r a n e , localised d i s t o r t i o n s o f t h e s p o r o z o i t e pellicle c o n c u r r e n t with active e n t r y o f the parasite, c o u l d result in the invagination o f h o s t l y m p h o c y t e plasmal e m m a . T h e s p o r o z o i t e s have b e e n s h o w n t o h a r b o u r i n t a c t f u n c t i o n a l m e t a b o l i c e n e r g y p a t h w a y s , and t h u s i n d e p e n d e n t l y generate the ATP req u i r e d f o r their invasive activities culminating in intracellular localisation (Jura et al., 1 9 8 4 ) . A n o t h e r m e c h a n i s m , i n d u c e d phagocytosis, has been suggested f o r T o x o p l a s m a gondii ( J o n e s et al., 1 9 7 2 ; R y n i n g and R e m i n g t o n , 1978). ACKNOWLEDGEMENTS I am i n d e b t e d t o the D i r e c t o r o f the CVTM, Professor D.W. B r o c k l e s b y f o r the provision o f facilities t h a t m a d e it possible t o u n d e r t a k e this s t u d y and f o r e n c o u r a g e m e n t . M y t h a n k s are d u e t o the D i r e c t o r o f the V R D , Dr. W.N. Masiga, f o r giving me permission t o carry o u t the investigation and f o r e n c o u r a g e m e n t . I am also grateful t o C.G.D. B r o w n and A.C. R o w l a n d for guidance and e n c o u r a g e m e n t during the study. My t h a n k s are d u e t o Dr. A.R. Walker for providing i n f e c t e d ticks, Mr. B. Kelly f o r t e c h n i c a l assistance and P a m e l a J u r a for t y p i n g the m a n u s c r i p t . This w o r k was f u n d e d as part o f a p r o j e c t s u p p o r t e d b y t h e Overseas D e v e l o p m e n t A d m i n i s t r a t i o n . Dr. W.G.Z.O. J u r a was s p o n s o r e d b y the British Council and this s t u d y cons t i t u t e d part o f his Ph.D. Thesis p r o j e c t . REFERENCES Brown, C.G.D., 1979. Propagation of Theileria. In: K. Maramorosch and H. Hirumi (Editors), Practical Applications of Tissue Culture. Academic Press, New York, pp. 223--245. Danforth, H.D., Aikawa, M., Cochrane, A.H. and Nussenzweig, R.S., 1980. Sporozoites of mammalian malaria: attachment to, interiorisation and fate within macrophages. J. Protozool., 27: 193--202. Fawcett, D.W., Doxsey, S., Stagg, D.A. and Young, A.S., 1982. The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Electron microscopic observations. Eur. J. Cell Biol., 27 : 10--21. Fletcher, J.M., Greenfield, B.F., Hardy, C.J., Scargill, D. and Woodhead, J.L., 1961. Ruthenium red. J. Chem. Soc., 2000--2006. Jensen, J.B. and Hammond, D.M., 1975. Ultrastructure of the invasion of Eimeria magna sporozoites into cultured cells. J. Protozool., 22: 411--415. Jensen, J.B. and Edgar, S.A., 1976. Possible secretory function of the rhoptries of Eimeria magna during penetration of cultured cells. J. Protozool., 62: 988--992. Jones, T.C., Yeh, S. and Hirsch, J.G., 1972. The interaction between Toxoplasma gondii and mammalian cells. I. Mechanism of interaction and intracellular fate of the parasite. J. Exp. Med., 136: 1157--1172. Jura, W.G.Z.O., 1981. Light and electron microscopic study of bovine lymphocyte penetration by Theileria annulata sporozoites in vitro. Trans R. Soc. Trop. Med. Hyg., 75: 898.
286 Jura, W.G.Z.O., 1983. Ultrastructural observations on host--parasite relationships of Theileria annulata and Theileria parva in vitro. Ph.D. Thesis, University of Edinburgh, Scotland. Jura, W.G.Z.O., Brown, C.G.D. and Kelly, B., 1983. Fine structure and invasive behaviour of the early developmental stages of Theileria annulata in vitro. Vet. Parasitol., 12: 31--44. Jura, W.G.Z.O., Brown, C.G.D. and Broeklesby, D.W., 1984. Metabolic energy pathways in Theileria annulata sporozoites and their significance in sporozoite--bovine lymphocyte interactions in vitro. Vet. Parasitol., 17: 000--000. Luft, J.H., 1971. Ruthenium red and violet. I. Chemistry, purification, methods of use for electron microscopy and mechanisms of action. Anat. Rec., 171 : 347--368. Mangin, L., 1983. Sur l'emploi du rouge de ruthenium en anatomie v~g~tale. C.R. Acad. Sci., 116: 653--656. Metais, M. and Schrevel, J., 1974. Quelques aspects du m~canisme de la motilit~ du protozoaire Selenidium hollandei. J. Protozool. Suppl., 473. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. Biol., 17: 208--212. Russel, D.G., 1983. Host cell invasion by Apicomplexa: an expression of the parasite's contractile system? Parasitology, 87 : 199--209. Ryning, F.W. and Remington, J.S., 1978. Effect of cytochalasin D at Toxoplasma gondii cell entry. Infect. Immun., 20: 739--743. Watson, M.L., 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475--478.
279
THE INTEGRITY OF HOST LYMPHOCYTE PLASMALEMMA DURING ITS I N V A S I O N BY T H E I L E R I A A N N U L A T A S P O R O Z O I T E S AS D E M O N S T R A T E D BY R U T H E N I U M R E D S T A I N I N G
W.G.Z.O. JURA Veterinary Research Department, K.A.R.I., Muguga, P.O. Box 32, Kikuyu (Kenya) (Accepted 26 October 1984)
ABSTRACT Jura, W.G.Z.O., 1985. The integrity of host lymphocyte plasmalemma during its invasion by Theileria annulata sporozoites as demonstrated by ruthenium red staining. Vet. Parasitol., 17: 279--286. The integrity of host lymphocyte plasma membrane during its invasion by Theileria annulata sporozoites iwas examined in an in vitro system using ruthenium red staining. It was demonstrated that during interiorisation of the sporozoites, the dye was completely excluded from the intracellular milieu, indicating that the continuity of the host-ceU plasmalemma was not disrupted throughout the process of penetration, and confirmed a previous observation that the fragmentation of the host lymphocyte membrane only occurs later, after the parasite is intracellular. INTRODUCTION Previous s t u d i e s have s h o w n t h a t Theileria annulata (Jura, 1981; J u r a et al., 1 9 8 3 ) and T. parva ( F a w c e t t et al., 1 9 8 2 ) s p o r o z o i t e s interiorise b y d e e p e n i n g an invagination o f the target b o v i n e - l y m p h o c y t e m e m b r a n e . T h e host-cell m e m b r a n e investing the interiorised s p o r o z o i t e f r a g m e n t s within 30 min o f e n t r y so t h a t the parasite remains in direct c o m m u n i c a t i o n with the c y t o s o l o f the h o s t l y m p h o c y t e ( J u r a et al., 1983). W h e t h e r some damage o c c u r s t o the h o s t l y m p h o c y t e p l a s m a l e m m a during the e n t r y o f sporozoites has n o t b e e n conclusively d e t e r m i n e d . In this s t u d y , the integrity o f the h o s t l y m p h o c y t e m e m b r a n e during the interiorisation o f T. annulata s p o r o z o i t e s was e x a m i n e d using r u t h e n i u m red staining. T h e m e c h a n i s m o f a c t i o n o f r u t h e n i u m red (Mangin, 1 9 8 3 ) , [RU302(NH3)14] C16"4H20 ( F l e t c h e r et al., 1961) was studied and discussed b y L u f t (1971). T h e d y e binds to extracellular acid m u c o p o l y s a c c h a r i d e s and related substances with o s m i u m t e t r o x i d e (OsO4) providing general f i x a t i o n and low c o n t r a s t so t h a t brownish-black deposits localise at sites w h e r e t h e r u t h e n i u m red--OsO4-coupled r e a c t i o n occurs. T h e first e v e n t is t h e linking o f t h e
0304-4017/85/$03.30
© 1985 Elsevier Science Publishers B.V.
280 ruthenium red to the acidic polysaccharides of tissues, whereby the specificity of the reaction is expressed. The ruthenium red is then oxidised by OsO4 in the mixture of ruthenium brown which in turn oxidises the polysaccharide to which it is bound. The overall reaction is an oxidation of the polysaccharide with an equivalent reduction of OsO4 to lower insoluble oxidation products which are localised extracellularly. The exclusion of ruthenium red--OsO4 reaction products delineates cell membranes and permits tracing tortuous plasmalemmal invaginations so that no intracellular staining is possible unless the continuity of the cell membrane is disrupted. MATERIALS AND METHODS
Preparation o f T. annulata sporozoite and peripheral blood lymphocyte (PBL ) suspensions and establishment o f cultures Details of the methods, modified after Brown (1979), by which PBL were separated from bovine venous blood, and by which sporozoites of T. annulata were prepared from infected ticks, have been described elsewhere (Jura et al., 1983). In brief, 3-day.fed Hyalomma anatolicum anatolicum ticks infected with T. annulata were surface-sterilised and ground in 3.5% bovine plasma albumin (Armour fraction V, Sigma Chemical Company, St. Louis, Missouri) in Eagle's minimum essential medium (MEM) (Gibco--Europe) to provide ground up tick supernate (GUTS) of a concentration equivalent to 2 ticks m1-1 and filtered to 8/1 (GUTS filtrate). PBL suspension was prepared from b u f f y coat obtained from defibrinated blood and layered on to a Ficoll/sodium diatrizoate gradient (Ficoll-plaque, Pharmacia Fine Chemicals, Uppsala, Sweden). The cell concentration was adjusted to 8 × 106 cells ml -~ of growth medium-RPMI 1640 with 20% foetal calf serum (Gibco--Europe), containing 100 i.u. m1-1 benzylpenicillin, 100 tlg ml -~ streptomycin sulphate (Glaxo Laboratories Limited, Greenford, England) and 2 mM ml -~ L-glutamine (Gibco-Europe). Cultures were established in triplicate by mixing 0.25 ml GUTS filtrate with 0.25 ml PBL suspension. Plates containing cultures were placed in a humidified box, gassed with 5% CO2/95% air and incubated at 37°C.
Preparation o f stock solution o f ruthenium red Commercial ruthenium red (50 mg) (John Mathey and Company Inc., 608 Fifth Avenue, New York, U.S.A.) was weighed, crushed in a mortar and a few drops of deionised, distilled water were added while continuing the crushing. A total of 5 ml distilled water was added to make 10 mg m1-1 final concentration, and the suspension transferred to a 15-ml centrifuge tube and heated in a water bath at 60°C with frequent agitation for 5 min then centrifuged at 1500 g for 10 min at 20°C (3000 r. min -1, MSE Chilspin). The supernate was stored at 4°C as a stock solution.
281
Procedure for ruthenium red staining The ruthenium red technique was applied to samples obtained at 15, 30 and 60 min of incubation to the technique described by Luft (1971). Suspensions of cells were centrifuged at 275 g for 10 min at 20°C {1450 r. min -~, MSE Minor), and pellets fixed for 1 h at 4°C in a freshly-prepared mixture of 0.5 ml 0.2 M cacodylate-buffered glutaraldehyde, 0.5 ml 0.2 M cacodylate buffer, pH 7.4 and 0.5 ml stock solution of ruthenium red. The pellets were then rinsed in 3 changes of 0.2 M cacodylate buffer over a period of 10 min and fixed again at 20°C for 3 h in freshly-prepared mixture of 0.5 ml 5% OsO4 in distilled water, 0.5 ml 0.2 M cacodylate buffer and 0.5 ml stock solution of ruthenium red. They were then washed in 3 changes of 0.2 M
Fig. 1. A non-infected ~bovine lymphocyte fixed and post fixed in glutaraldehyde-ruthenium red and OsO~--ruthenium red mixtures, respectively, and processed to araldite by routine electron microscopic techniques. Complete localisation of reaction products of ruthenium red, with distinct delineation of lymphocyte plasmalemma is demonstrated by increased electron density ( ~ ' ) of the membrane. Magnification, X 20 87
282 c a c o d y l a t e buffer, d e h y d r a t e d in graded series o f e t h a n o l and p r o c e s s e d to araldite. Sections, c u t w i t h L K B glass knives on a C a m b r i d g e - H u x l e y Ultram i c r o t o m e , w e r e m o u n t e d on c o p p e r grids, stained w i t h u r a n y l a c e t a t e (Watson, 1 9 5 8 ) and lead citrate ( R e y n o l d s , 1 9 6 3 ) and e x a m i n e d on a Philips 400 electron microscope. RESULTS C o m p l e t e e x t r a c e l l u l a r localisation o f r e a c t i o n p r o d u c t s o f r u t h e n i u m red, w i t h distinct d e l i n e a t i o n of a n o n - i n f e c t e d l y m p h o c y t e p l a s m a l e m m a was d e m o n s t r a t e d b y increased e l e c t r o n d e n s i t y o f t h e m e m b r a n e (Fig. 1). O b s e r v a t i o n s d u r i n g t h e various stages o f i n t e r i o r i s a t i o n o f T. a n n u l a t a
Fig. 2. A T. annulata sporozoite in the process of interiorisation and stained with ruthenium red is illustrated. The target lymphocyte plasmalemma is deeply invaginated so that its ends (.~,,m) are close to fusing around the invading parasite. The lymphocyte membrane circumjacent to the sporozoite ( ~ ) remains intact throughout the process as demonstrated by lack of intracellular ruthenium red staining. A portion of host nucleus ( HN ) is shown. Magnification, x 128 590.
283 sporozoites (Figs. 2 and 3) showed that the cell membrane circumjacent to the invading parasite remained intact so that no intraceUular ruthenium red--OsO4 reaction products were observed. In cases where the sporozoites had been completely interiorised, and the invaginated host membrane fused and sealed before staining (Fig. 4), so t h a t there was no communication with extracellular milieu, no ruthenium red was demonstrated in the host plasma membrane investing the parasite. DISCUSSION In this study, it has been demonstrated by ruthenium red staining that when T. annulata sporozoites invade and enter bovine lymphocytes, the dye is completely excluded from the intracellular milieu, indicating that the continuity of the plasmalemma of the host cell is not disrupted, and remains
Fig. 3. A T. annulata sporozoite processed with ruthenium red-fixative mixtures in the course of interiorisation is almost completely intracellular. The ends ( ~ ) of the invaginating target l y m p h o c y t e membrane are touching, but not yet fused. The investing host cell membrane as well as parasite pellicle are delineated by electron-dense ruthenium red--OsO 4 reaction products ( ~ = : ) . No reaction products are intracellular, coni~nTning that T. annulata sporozoites do not disrupt target cell membrane during the entry process. Magnification, × 115 500.
284 intact throughout the process of interiorisation. However, a short while after the parasite is intracellular, the circumjacent host membrane has been shown to break up and disappear (Jura et al., 1983). Similarly, certain members of the phylum Apicomplexa such as Selenidium hollandei (Metals ~nd Schrevel, 1974), Eimeria magna (Jensen and Hammond, 1975; Jensen and Edgar, 1976) and mammalian malaria parasites, Plasmodium berghei and P. knowlesi (Danforth et al., 1980) have also been shown to achieve interiorisation by active invasion and invagination of the host-cell membrane without disrupting its continuity. H o w the parasites accomplish the invagination of their host membranes is no t clear, but several suggestions have been made. Russel (1983) suggested that Eimeria tenella and E. acervulina enter the parasitophorous vacuole by "capping" the host/parasite junction down their body, so locomoting into the host cell. He believed the mechanism required no additional modifications to either cell, was facilitated by the specificity of
Fig. 4. An electron micrograph s h o w i n g intracellular and attached T. annulata sporozoites processed with r u t h e n i u m red--fixative mixture. The intracellulax parasite interiorised prior to r u t h e n i u m red treatment hence no reaction products axe demontsrated either on its pellicle ( P ) or on the circumjaeent host membrane. The attached sporozoite and the l y m p h o c y t e membranes are densely covered with r u t h e n i u m red--OsO 4 reaction products (,qJ====). Magnification, x 43 250.
285 m e m b r a n e r e c e p t o r s , and the parasite's c o n t r a c t i l e s y s t e m was the p r i m e " m o v e r " . J u r a ( 1 9 8 3 ) o b s e r v e d t h a t following a firm r e c e p t o r - d e p e n d e n t a t t a c h m e n t o f T. annulata s p o r o z o i t e s t o t a r g e t l y m p h o c y t e m e m b r a n e , localised d i s t o r t i o n s o f t h e s p o r o z o i t e pellicle c o n c u r r e n t with active e n t r y o f the parasite, c o u l d result in the invagination o f h o s t l y m p h o c y t e plasmal e m m a . T h e s p o r o z o i t e s have b e e n s h o w n t o h a r b o u r i n t a c t f u n c t i o n a l m e t a b o l i c e n e r g y p a t h w a y s , and t h u s i n d e p e n d e n t l y generate the ATP req u i r e d f o r their invasive activities culminating in intracellular localisation (Jura et al., 1 9 8 4 ) . A n o t h e r m e c h a n i s m , i n d u c e d phagocytosis, has been suggested f o r T o x o p l a s m a gondii ( J o n e s et al., 1 9 7 2 ; R y n i n g and R e m i n g t o n , 1978). ACKNOWLEDGEMENTS I am i n d e b t e d t o the D i r e c t o r o f the CVTM, Professor D.W. B r o c k l e s b y f o r the provision o f facilities t h a t m a d e it possible t o u n d e r t a k e this s t u d y and f o r e n c o u r a g e m e n t . M y t h a n k s are d u e t o the D i r e c t o r o f the V R D , Dr. W.N. Masiga, f o r giving me permission t o carry o u t the investigation and f o r e n c o u r a g e m e n t . I am also grateful t o C.G.D. B r o w n and A.C. R o w l a n d for guidance and e n c o u r a g e m e n t during the study. My t h a n k s are d u e t o Dr. A.R. Walker for providing i n f e c t e d ticks, Mr. B. Kelly f o r t e c h n i c a l assistance and P a m e l a J u r a for t y p i n g the m a n u s c r i p t . This w o r k was f u n d e d as part o f a p r o j e c t s u p p o r t e d b y t h e Overseas D e v e l o p m e n t A d m i n i s t r a t i o n . Dr. W.G.Z.O. J u r a was s p o n s o r e d b y the British Council and this s t u d y cons t i t u t e d part o f his Ph.D. Thesis p r o j e c t . REFERENCES Brown, C.G.D., 1979. Propagation of Theileria. In: K. Maramorosch and H. Hirumi (Editors), Practical Applications of Tissue Culture. Academic Press, New York, pp. 223--245. Danforth, H.D., Aikawa, M., Cochrane, A.H. and Nussenzweig, R.S., 1980. Sporozoites of mammalian malaria: attachment to, interiorisation and fate within macrophages. J. Protozool., 27: 193--202. Fawcett, D.W., Doxsey, S., Stagg, D.A. and Young, A.S., 1982. The entry of sporozoites of Theileria parva into bovine lymphocytes in vitro. Electron microscopic observations. Eur. J. Cell Biol., 27 : 10--21. Fletcher, J.M., Greenfield, B.F., Hardy, C.J., Scargill, D. and Woodhead, J.L., 1961. Ruthenium red. J. Chem. Soc., 2000--2006. Jensen, J.B. and Hammond, D.M., 1975. Ultrastructure of the invasion of Eimeria magna sporozoites into cultured cells. J. Protozool., 22: 411--415. Jensen, J.B. and Edgar, S.A., 1976. Possible secretory function of the rhoptries of Eimeria magna during penetration of cultured cells. J. Protozool., 62: 988--992. Jones, T.C., Yeh, S. and Hirsch, J.G., 1972. The interaction between Toxoplasma gondii and mammalian cells. I. Mechanism of interaction and intracellular fate of the parasite. J. Exp. Med., 136: 1157--1172. Jura, W.G.Z.O., 1981. Light and electron microscopic study of bovine lymphocyte penetration by Theileria annulata sporozoites in vitro. Trans R. Soc. Trop. Med. Hyg., 75: 898.
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