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In vitro propagation of Cytoecetes phagocytophila, the causative agent of tick-borne fever
Veterinary Microbiology, 7 (1982) 127--133 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
127
IN VITRO PROPAGATION OF CYTOECETES PHAGOCYTOPHILA, THE CAUSATIVE AGENT OF TICK-BORNE FEVER
Z. WOLDEHIWET and G.R. SCOTT
Centre for Tropical Veterinary Medicine, University of Edinburgh, Roslin, Midlothian EH25 9RG (Great Britain) (Accepted 26 October 1981)
ABSTRACT
Woldehiwet, Z. and Scott, G.R., 1982. In vitro propagation of Cytoecetes phagocytophila, the causative agent of tick-borne fever. Vet. Microbiol., 7:127--133.
Cytoecetes phagocytophila, a neutrophilic rickettsia which parasitizes sheep and cattle, was propagated transiently at 37°C in cultures of heparinized whole blood of sheep supplemented with Medium 199 containing HEPES buffer. Significant increases in the number of infected cells and the number of rickettsias per infected cell were observed within 24 h. Back-passage into sheep produced typical tick-borne fever. INTRODUCTION T i c k - b o r n e fever was f o r t u i t o u s l y d i s c o v e r e d as a disease o f s h e e p on ticki n f e s t e d p a s t u r e s o f S c o t l a n d b y M a c L e o d ( 1 9 3 2 ) while investigating t h e tickt r a n s m i s s i o n o f louping-ill. G o r d o n et al. ( 1 9 4 0 ) d e m o n s t r a t e d t h e causal o r g a n i s m in t h e g r a n u l a r l e u c o c y t e s a n d m o n o c y t e s o f i n f e c t e d s h e e p a n d s p e c u l a t e d t h a t t h e infective a g e n t was p r o b a b l y a rickettsia. Foggie ( 1 9 5 1 ) suggested t h a t t h e o r g a n i s m s h o u l d be classified w i t h t h e rickettsias as Rickettsia phagocytophila, o n t h e basis o f its intracellular locat i o n , its a r t h r o p o d v e c t o r a n d its m o r p h o l o g i c a l r e s e m b l a n c e t o w h a t are n o w k n o w n as Chlamydia. This classification was later r e j e c t e d o n t h e g r o u n d s t h a t t h e o r g a n i s m h a d little r e s e m b l a n c e t o t r u e r i c k e t t s i a s such as t h o s e causing t y p h u s a n d R o c k y - M o u n t a i n s p o t t e d f e v e r (Foggie, 1 9 6 2 ) . H e also r e j e c t e d a n y a s s o c i a t i o n w i t h t h e Chlamydia b e c a u s e h e did n o t find a n y serological or i m m u n o l o g i c a l c o n n e c t i o n b e t w e e n t h e a g e n t o f t i c k - b o r n e f e v e r and t h e Chlamydia causing e n z o o t i c a b o r t i o n in ewes (Foggie, 1962). O n t h e o t h e r h a n d , he a r g u e d t h a t t h e o r g a n i s m causing t i c k - b o r n e fever h a d striking m o r p h o l o g i c a l similarities w i t h Cytoecetes microti, an o r g a n i s m f o u n d in t h e p o l y m o r p h o n u c l e a r cells o f t h e vole ( T y z z e r , 1 9 3 8 ) . Foggie, t h e r e f o r e , p r o p o s e d t h a t t h e causative a g e n t o f t i c k - b o r n e f e v e r s h o u l d be i n c l u d e d in t h e genus c r e a t e d b y T y z z e r ( 1 9 3 8 ) as Cytoecetesphagocytophila in t h e t r i b e Ehrlichieae t o distinguish it f r o m t h e genus Ehrlichia o f t h e s a m e tribe, t h e
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128 latter being reserved for those animal rickettsias which parasitize m ononuclear leucocytes. Kraus et al. (1972) added the causative agent o f bovine petechial fever to the same genus as Cytoecetes ondiri. Cytoecetes phagocytophila has not, hitherto, been propagated in ovo or in vitro (Tuomi, 1967; Thrusfield et al., 1978). The failure to propagate C. phagocytophila in vitro has been attributed to the difficulty of culturing the target cells (Thrusfield et al., 1978). We r e p o r t herein a simple m e t h o d of propagating the organism in whole blood cultures. MATERIALS AND METHODS
Cy toece tes phagocy toph ila A stabilate o f the Old Sourhope (OS) strain originally isolated by Foster and Cameron (1970) was used. The stabilate was kept at -114°C as heparinized infected blood with 10% di m et hyl sulphoxide.
Experimental infection Adult sheep obtained from tick-free areas were infected by an intravenous inoculation o f 1 ml of a 10 -1 dilution of the stabilate in phosphate-buffered saline at pH 7.2. Blood was collected f r om infected and non-infected sheep in sterile containers coated with heparin estimated to give a final concent rat i on of 10 I.U. o f heparin per ml of blood.
Whole blood cultures Culture medium. Medium 199 with Earle's salts, 25 mM HEPES buffer and L-glutamine was commercially supplied. Ten percent foetal calf serum and 100 I.U. o f penicillin per ml were added immediately before use.
Culture techniques. In one series of experiments 1 ml o f infected blood was mixed with 3 ml of medium in disposable polystyrene bottles. The bottles were left at 37°C either stationary or under continuous agitation for 4, 8 or 24 h. Controls were left at 4°C for 24 h. In a second series of experiments 0.1 ml of infected blood was mixed with 0.9 ml of blood collected from susceptible sheep and 3 ml of medium and incubated at 37°C for 24 h. In a third series o f experiments 0.5 ml of plasma f r om infected blood was added t o 1 ml o f blood from susceptible sheep and incubated at 37°C for 24 h.
Sub-culture. To 0.9 ml of blood collected from susceptible sheep were added 0.1 ml o f infected blood cultured for 24 h and 3 ml of medium and the mixture incubated at 37°C for 24--48 h. Up to four blind passages were carried out.
129
Enumeration. L e u c o c y t e s w e r e c o u n t e d e l e c t r o n i c a l l y ( C o u l t e r E l e c t r o n i c s Ltd.). I n f e c t i o n rates w e r e e s t i m a t e d f r o m G i e m s a - s t a i n e d s m e a r s o f b l o o d b e f o r e a n d a f t e r culture. W h e n the n u m b e r o f i n f e c t e d cells was low, acridine o r a n g e stain was used. Cells c o n t a i n i n g o n e or m o r e inclusions w e r e r e c o r d e d as i n f e c t e d ; i n f e c t i o n r a t e s w e r e e x p r e s s e d as p e r c e n t a g e s o f t o t a l b l o o d leucocytes.
Titration of infectivity A t t e m p t s t o assess d i f f e r e n c e s in t h e i n f e c t i v i t y o f fresh i n f e c t e d b l o o d a n d i n f e c t e d b l o o d c u l t u r e d f o r 24 h w e r e m a d e b y a r o u g h t i t r a t i o n o f inf e c t e d b l o o d using o n e s h e e p p e r dilution. S u s c e p t i b l e s h e e p w e r e i n o c u l a t e d w i t h serial dilutions o f i n f e c t e d b l o o d b e f o r e a n d a f t e r 24 h o f culture.
Analysis of data Means a n d s t a n d a r d d e v i a t i o n s w e r e c a l c u l a t e d a c c o r d i n g to c o n v e n t i o n a l m e t h o d s . Pair-differences w e r e a n a l y s e d b y S t u d e n t ' s t-test. RESULTS W h e n b l o o d f r o m i n f e c t e d s h e e p was i n c u b a t e d at 37°C w i t h H E P E S - b u f f e r e d m e d i u m 199 t h e n u m b e r o f i n f e c t e d cells increased w i t h i n 4 h (Table I). I n c u b a t i o n o f i n f e c t e d cells f o r 8 or 24 h r e s u l t e d in f u r t h e r increases while c o n t r o l s left at 4°C f o r 24 h did n o t s h o w a n y increases (Table I). T h e perc e n t a g e s o f i n f e c t e d cells a f t e r 4 h o f c u l t u r e w e r e significantly higher t h a n t h o s e o f fresh b l o o d s a m p l e s (ts = 3.75; P < 0.010). Similarly t h e n u m b e r o f i n f e c t e d cells o b t a i n e d a f t e r 8 a n d 24 h o f c u l t u r e was signific a n t l y higher t h a n t h o s e in fresh s a m p l e s (t8 = 4.70; P < 0 . 0 1 0 and t23 = 11.21; P < 0 . 0 0 1 r e s p e c t i v e l y ) . In c o n t r a s t , t h e p e r c e n t a g e s o f i n f e c t e d cells o f b l o o d s a m p l e s left at 4°C f o r 24 h w e r e significantly less t h a n t h o s e in fresh s a m p l e s (ts = 2.77; P < 0.50). TABLE I Means and standard deviations of percentages of infected cells before and after culture Hours at 37°C
Mean
Standard deviation
n
0 4 8 24 24 a
22.79 33.22 42.44 47.83 15.10
9.27 15.53 17.41 11.70 10.24
24 9 9 24 9
aAt 4°C.
130
There were no significant differences between the percentages o f infected cells in samples cultured for 8 h and 24 h (t8 = 0.68; P < 0.50) but the differences b etween the percentages of infected cells in samples obtained after 4 h and 8 h o f culture were significant (t8 = 3.47; P < 0.020). There were no significant differences in the numbers of infected cells in samples left under c o n t in u o u s agitation and those left stationary (t8 = 0.14; P > 0.90). When infected blood was mixed with blood collected f r o m susceptible sheep and cultured for 24 h, statistically significant increases in t he percentages o f infected cells occurred (t18 = 3.38; P < 0.010) but when the plasma f r o m infected sheep was added to normal uni nfect ed blood no infected cells were observed after 24 h or 48 h of culture. Three successive subcultures were achieved on f our occasions but all died o ut after the third passage. A susceptible sheep inoculated with a second passage reacted positively and typical inclusions of C. phagocy tophila were d e m o n s t r a t e d in the granulocytes. Increases in the numbers o f organisms per infected cell as well as marked changes in the p r e d o m i n a n t morphological types were observed following culture. F r o m 13 infected blood samples studied there were means of 45.23 +_ 11..44 discrete particles per 20 infected cells and 3.15 +- 4.95 clusters per 20 infected cells. After culturing infected blood for 24 h there were 60.69 + 31.15 discrete particles and 28.92 -+ 12.80 clusters per 20 infected cells. The increases in discrete particles and clusters after culture w e r e significant (t,: = 6.42; P < 0.001 and t,2 = 2.34; P < 0.050 respectively). Rough titrations did n o t reveal significant differences between the infectivity levels of fresh and cultured blood samples (Table II). Out of four samples tested only one sample had higher infectivity titre after culture. TABLE II Infectivity titres (IDs0) of infected blood before and after culture Sample
Fresh
Cultured
120 158 165 166
10 s 106 104 i0 ~
107 106 104 106
DISCUSSION
Thrusfield et al. {1978) attributed past failures to propagate Cophagocytophila to the fact tha t the cells pr e dom i nant l y parasitized by the organism were the granular leucocytes which have not y e t been cultured in vitro. Indeed, we f o u n d in this study that the leucocytes started to degenerate very rapidly, the total counts, as estimated by an electronic counter, being reduced significantly within 24 h. Despite this, however, the organisms c o n t i n u e d to
131
Fig. 1. Culture of infected blood. (A) Infected leucocyte packed with organisms after 24 h of culture. (B) Clusters of organisms with no host cell after 24 h.
132 m u l t i p l y in a p p a r e n t l y d e g e n e r a t i n g cells a n d on m a n y occasions g r o u p s o f o r g a n i s m s were f o u n d w i t h little or n o h o s t cell (Fig. 1). Ristic ( 1 9 7 6 ) suggested t h a t the ability o f t h e h o s t cell to r e p l i c a t e is an essential r e q u i r e m e n t f o r t h e d e v e l o p m e n t o f c u l t u r e s y s t e m s f o r a n i m a l rickettsias w h i c h parasitize l e u c o c y t e s . H e cited the success o f the p r o p a g a t i o n o f E h r l i c h i a c a n i s in m o n o c y t i c cultures ( N y i n d o et al., 1971). Weiss ( 1 9 7 3 ) , o n the o t h e r h a n d , t h o u g h t t h a t m u l t i p l i c a t i o n o f h o s t cells was n o t n e c e s s a r y f o r r i c k e t t s i a l g r o w t h . T h e increase in t h e p e r c e n t a g e o f i n f e c t e d cells w h e n i n f e c t e d b l o o d was m i x e d w i t h b l o o d f r o m s u s c e p t i b l e sheep suggested t h a t n e w cells :had b e e n invaded. O n t h e o t h e r h a n d , w h e n p l a s m a f r o m i n f e c t e d s h e e p was m i x e d w i t h b l o o d f r o m s u s c e p t i b l e s h e e p no i n f e c t e d cells w e r e o b s e r v e d a f t e r culture. T h e l a t t e r finding suggests t h a t e i t h e r t h e cell-free f o r m was t o o fragile t o initiate i n f e c t i o n in v i t r o or t h e increases in t h e n u m b e r s o f i n f e c t e d cells w e r e n o t d u e t o n e w cells being i n v a d e d b u t d u e t o t h e c o n t i n u e d m u l t i p l i c a t i o n o f t h e o r g a n i s m s in a l r e a d y i n f e c t e d cells. T h e t e c h n i q u e d e s c r i b e d h e r e p r o v i d e s a m e a n s f o r growin~ t h e o r g a n i s m in s u f f i c i e n t q u a n t i t i e s f o r d e t a i l e d studies o f its p h y s i c a l a n d c h e m i c a l p r o p e r ties a n d f o r i m p r o v i n g antigen yields f o r serological a n d i m m u n o l o g i c a l studies. T h e ability o f the o r g a n i s m t o c o n t i n u e t o p r o p a g a t e in vitro f o r at least 24 h gives a h i t h e r t o i m p o s s i b l e p r o c e d u r e f o r s t u d y i n g t h e m o r p h o l o g i c a l c h a n g e s t h a t o c c u r w i t h i n the i n f e c t e d cell. P r e l i m i n a r y in vitro studies in this d i r e c t i o n indicate t h a t t h e r e is a s e q u e n c e o f d e v e l o p m e n t f r o m single discrete particles to clusters o f particles. ACKNOWLEDGEMENT T h e w o r k was financially s u p p o r t e d , in p a r t , b y t h e Overseas D e v e l o p m e n t Administration, London.
REFERENCES
Foggie, A., 1951. Studies on the infectious agent of tick-borne fever in sheep. J. Pathoi. Bacteriol., 63: 1--15. Foggie, A., 1962. Studies on tick pyaemia and tick-borne fever. Syrup. Zool. Soc. London, 6: 51--60. Foster, W.N.M. and Cameron, A.E., 1970. Observations on ovine strains of tick-borne fever. J. Comp. Pathol., 80: 429--436. Gordon, W.S., Brownlee, A. and Wilson, D.R., 1940. Studies in louping ill, tick-borne fever and scrapie. Proc. 3rd Int. Conf. Microbiol., New York, pp. 362--363. Kraus, H., Davies, F.G., Qdegaard, O.A. and Cooper, J.E., 1972. The morphology of the causative agent of bovine petechial fever (Ondiri disease). J. Comp. Pathol., 82: 241-246. MacLeod, J., 1932. Preliminary studies in the tick transmission of louping ill. II. A study of the reaction of sheep to tick infestation. Vet. J., 88: 276--284. NyindO, M.B.A., Ristic, M., Huxsoll, D.L. and Smith, A.R., 1971. Tropical canine pancytopaenia -- in vitro cultivation of the causative agent, Ehrlichia canis. Am. J. Vet. Res., 32: 1651--1658.
133 Ristic, M., 1976. Immunologic systems and protection of infections caused by intra-cellular blood parasites. Vet. Parasitol., 2: 31--47. Tuomi, J., 1967. Experimental studies on bovine tick-borne fever (1) Clinical and haematological data, some properties of the causative agent and homologous immunity. Acta Pathol. Mierobiol. Scand., 70: 429--445. Thrusfield, M.V., Synge, B.A. and Scott, G,R., 1978. Attempts to cultivate Ehrlichia phagocytophila in vitro. Vet. Microbiol., 2: 257--260. Tyzzer, E.E., 1938. Cytoecetes microti, N.G.N. Sp., a parasite developing in granulocytes and infective to small rodents. Parasitology, 30: 242--257. Weiss, E., 1973. Growth and physiology of Rickettsieae. Bacteriol. Rev., 37: 259--283.
127
IN VITRO PROPAGATION OF CYTOECETES PHAGOCYTOPHILA, THE CAUSATIVE AGENT OF TICK-BORNE FEVER
Z. WOLDEHIWET and G.R. SCOTT
Centre for Tropical Veterinary Medicine, University of Edinburgh, Roslin, Midlothian EH25 9RG (Great Britain) (Accepted 26 October 1981)
ABSTRACT
Woldehiwet, Z. and Scott, G.R., 1982. In vitro propagation of Cytoecetes phagocytophila, the causative agent of tick-borne fever. Vet. Microbiol., 7:127--133.
Cytoecetes phagocytophila, a neutrophilic rickettsia which parasitizes sheep and cattle, was propagated transiently at 37°C in cultures of heparinized whole blood of sheep supplemented with Medium 199 containing HEPES buffer. Significant increases in the number of infected cells and the number of rickettsias per infected cell were observed within 24 h. Back-passage into sheep produced typical tick-borne fever. INTRODUCTION T i c k - b o r n e fever was f o r t u i t o u s l y d i s c o v e r e d as a disease o f s h e e p on ticki n f e s t e d p a s t u r e s o f S c o t l a n d b y M a c L e o d ( 1 9 3 2 ) while investigating t h e tickt r a n s m i s s i o n o f louping-ill. G o r d o n et al. ( 1 9 4 0 ) d e m o n s t r a t e d t h e causal o r g a n i s m in t h e g r a n u l a r l e u c o c y t e s a n d m o n o c y t e s o f i n f e c t e d s h e e p a n d s p e c u l a t e d t h a t t h e infective a g e n t was p r o b a b l y a rickettsia. Foggie ( 1 9 5 1 ) suggested t h a t t h e o r g a n i s m s h o u l d be classified w i t h t h e rickettsias as Rickettsia phagocytophila, o n t h e basis o f its intracellular locat i o n , its a r t h r o p o d v e c t o r a n d its m o r p h o l o g i c a l r e s e m b l a n c e t o w h a t are n o w k n o w n as Chlamydia. This classification was later r e j e c t e d o n t h e g r o u n d s t h a t t h e o r g a n i s m h a d little r e s e m b l a n c e t o t r u e r i c k e t t s i a s such as t h o s e causing t y p h u s a n d R o c k y - M o u n t a i n s p o t t e d f e v e r (Foggie, 1 9 6 2 ) . H e also r e j e c t e d a n y a s s o c i a t i o n w i t h t h e Chlamydia b e c a u s e h e did n o t find a n y serological or i m m u n o l o g i c a l c o n n e c t i o n b e t w e e n t h e a g e n t o f t i c k - b o r n e f e v e r and t h e Chlamydia causing e n z o o t i c a b o r t i o n in ewes (Foggie, 1962). O n t h e o t h e r h a n d , he a r g u e d t h a t t h e o r g a n i s m causing t i c k - b o r n e fever h a d striking m o r p h o l o g i c a l similarities w i t h Cytoecetes microti, an o r g a n i s m f o u n d in t h e p o l y m o r p h o n u c l e a r cells o f t h e vole ( T y z z e r , 1 9 3 8 ) . Foggie, t h e r e f o r e , p r o p o s e d t h a t t h e causative a g e n t o f t i c k - b o r n e f e v e r s h o u l d be i n c l u d e d in t h e genus c r e a t e d b y T y z z e r ( 1 9 3 8 ) as Cytoecetesphagocytophila in t h e t r i b e Ehrlichieae t o distinguish it f r o m t h e genus Ehrlichia o f t h e s a m e tribe, t h e
0378-1135/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
128 latter being reserved for those animal rickettsias which parasitize m ononuclear leucocytes. Kraus et al. (1972) added the causative agent o f bovine petechial fever to the same genus as Cytoecetes ondiri. Cytoecetes phagocytophila has not, hitherto, been propagated in ovo or in vitro (Tuomi, 1967; Thrusfield et al., 1978). The failure to propagate C. phagocytophila in vitro has been attributed to the difficulty of culturing the target cells (Thrusfield et al., 1978). We r e p o r t herein a simple m e t h o d of propagating the organism in whole blood cultures. MATERIALS AND METHODS
Cy toece tes phagocy toph ila A stabilate o f the Old Sourhope (OS) strain originally isolated by Foster and Cameron (1970) was used. The stabilate was kept at -114°C as heparinized infected blood with 10% di m et hyl sulphoxide.
Experimental infection Adult sheep obtained from tick-free areas were infected by an intravenous inoculation o f 1 ml of a 10 -1 dilution of the stabilate in phosphate-buffered saline at pH 7.2. Blood was collected f r om infected and non-infected sheep in sterile containers coated with heparin estimated to give a final concent rat i on of 10 I.U. o f heparin per ml of blood.
Whole blood cultures Culture medium. Medium 199 with Earle's salts, 25 mM HEPES buffer and L-glutamine was commercially supplied. Ten percent foetal calf serum and 100 I.U. o f penicillin per ml were added immediately before use.
Culture techniques. In one series of experiments 1 ml o f infected blood was mixed with 3 ml of medium in disposable polystyrene bottles. The bottles were left at 37°C either stationary or under continuous agitation for 4, 8 or 24 h. Controls were left at 4°C for 24 h. In a second series of experiments 0.1 ml of infected blood was mixed with 0.9 ml of blood collected from susceptible sheep and 3 ml of medium and incubated at 37°C for 24 h. In a third series o f experiments 0.5 ml of plasma f r om infected blood was added t o 1 ml o f blood from susceptible sheep and incubated at 37°C for 24 h.
Sub-culture. To 0.9 ml of blood collected from susceptible sheep were added 0.1 ml o f infected blood cultured for 24 h and 3 ml of medium and the mixture incubated at 37°C for 24--48 h. Up to four blind passages were carried out.
129
Enumeration. L e u c o c y t e s w e r e c o u n t e d e l e c t r o n i c a l l y ( C o u l t e r E l e c t r o n i c s Ltd.). I n f e c t i o n rates w e r e e s t i m a t e d f r o m G i e m s a - s t a i n e d s m e a r s o f b l o o d b e f o r e a n d a f t e r culture. W h e n the n u m b e r o f i n f e c t e d cells was low, acridine o r a n g e stain was used. Cells c o n t a i n i n g o n e or m o r e inclusions w e r e r e c o r d e d as i n f e c t e d ; i n f e c t i o n r a t e s w e r e e x p r e s s e d as p e r c e n t a g e s o f t o t a l b l o o d leucocytes.
Titration of infectivity A t t e m p t s t o assess d i f f e r e n c e s in t h e i n f e c t i v i t y o f fresh i n f e c t e d b l o o d a n d i n f e c t e d b l o o d c u l t u r e d f o r 24 h w e r e m a d e b y a r o u g h t i t r a t i o n o f inf e c t e d b l o o d using o n e s h e e p p e r dilution. S u s c e p t i b l e s h e e p w e r e i n o c u l a t e d w i t h serial dilutions o f i n f e c t e d b l o o d b e f o r e a n d a f t e r 24 h o f culture.
Analysis of data Means a n d s t a n d a r d d e v i a t i o n s w e r e c a l c u l a t e d a c c o r d i n g to c o n v e n t i o n a l m e t h o d s . Pair-differences w e r e a n a l y s e d b y S t u d e n t ' s t-test. RESULTS W h e n b l o o d f r o m i n f e c t e d s h e e p was i n c u b a t e d at 37°C w i t h H E P E S - b u f f e r e d m e d i u m 199 t h e n u m b e r o f i n f e c t e d cells increased w i t h i n 4 h (Table I). I n c u b a t i o n o f i n f e c t e d cells f o r 8 or 24 h r e s u l t e d in f u r t h e r increases while c o n t r o l s left at 4°C f o r 24 h did n o t s h o w a n y increases (Table I). T h e perc e n t a g e s o f i n f e c t e d cells a f t e r 4 h o f c u l t u r e w e r e significantly higher t h a n t h o s e o f fresh b l o o d s a m p l e s (ts = 3.75; P < 0.010). Similarly t h e n u m b e r o f i n f e c t e d cells o b t a i n e d a f t e r 8 a n d 24 h o f c u l t u r e was signific a n t l y higher t h a n t h o s e in fresh s a m p l e s (t8 = 4.70; P < 0 . 0 1 0 and t23 = 11.21; P < 0 . 0 0 1 r e s p e c t i v e l y ) . In c o n t r a s t , t h e p e r c e n t a g e s o f i n f e c t e d cells o f b l o o d s a m p l e s left at 4°C f o r 24 h w e r e significantly less t h a n t h o s e in fresh s a m p l e s (ts = 2.77; P < 0.50). TABLE I Means and standard deviations of percentages of infected cells before and after culture Hours at 37°C
Mean
Standard deviation
n
0 4 8 24 24 a
22.79 33.22 42.44 47.83 15.10
9.27 15.53 17.41 11.70 10.24
24 9 9 24 9
aAt 4°C.
130
There were no significant differences between the percentages o f infected cells in samples cultured for 8 h and 24 h (t8 = 0.68; P < 0.50) but the differences b etween the percentages of infected cells in samples obtained after 4 h and 8 h o f culture were significant (t8 = 3.47; P < 0.020). There were no significant differences in the numbers of infected cells in samples left under c o n t in u o u s agitation and those left stationary (t8 = 0.14; P > 0.90). When infected blood was mixed with blood collected f r o m susceptible sheep and cultured for 24 h, statistically significant increases in t he percentages o f infected cells occurred (t18 = 3.38; P < 0.010) but when the plasma f r o m infected sheep was added to normal uni nfect ed blood no infected cells were observed after 24 h or 48 h of culture. Three successive subcultures were achieved on f our occasions but all died o ut after the third passage. A susceptible sheep inoculated with a second passage reacted positively and typical inclusions of C. phagocy tophila were d e m o n s t r a t e d in the granulocytes. Increases in the numbers o f organisms per infected cell as well as marked changes in the p r e d o m i n a n t morphological types were observed following culture. F r o m 13 infected blood samples studied there were means of 45.23 +_ 11..44 discrete particles per 20 infected cells and 3.15 +- 4.95 clusters per 20 infected cells. After culturing infected blood for 24 h there were 60.69 + 31.15 discrete particles and 28.92 -+ 12.80 clusters per 20 infected cells. The increases in discrete particles and clusters after culture w e r e significant (t,: = 6.42; P < 0.001 and t,2 = 2.34; P < 0.050 respectively). Rough titrations did n o t reveal significant differences between the infectivity levels of fresh and cultured blood samples (Table II). Out of four samples tested only one sample had higher infectivity titre after culture. TABLE II Infectivity titres (IDs0) of infected blood before and after culture Sample
Fresh
Cultured
120 158 165 166
10 s 106 104 i0 ~
107 106 104 106
DISCUSSION
Thrusfield et al. {1978) attributed past failures to propagate Cophagocytophila to the fact tha t the cells pr e dom i nant l y parasitized by the organism were the granular leucocytes which have not y e t been cultured in vitro. Indeed, we f o u n d in this study that the leucocytes started to degenerate very rapidly, the total counts, as estimated by an electronic counter, being reduced significantly within 24 h. Despite this, however, the organisms c o n t i n u e d to
131
Fig. 1. Culture of infected blood. (A) Infected leucocyte packed with organisms after 24 h of culture. (B) Clusters of organisms with no host cell after 24 h.
132 m u l t i p l y in a p p a r e n t l y d e g e n e r a t i n g cells a n d on m a n y occasions g r o u p s o f o r g a n i s m s were f o u n d w i t h little or n o h o s t cell (Fig. 1). Ristic ( 1 9 7 6 ) suggested t h a t the ability o f t h e h o s t cell to r e p l i c a t e is an essential r e q u i r e m e n t f o r t h e d e v e l o p m e n t o f c u l t u r e s y s t e m s f o r a n i m a l rickettsias w h i c h parasitize l e u c o c y t e s . H e cited the success o f the p r o p a g a t i o n o f E h r l i c h i a c a n i s in m o n o c y t i c cultures ( N y i n d o et al., 1971). Weiss ( 1 9 7 3 ) , o n the o t h e r h a n d , t h o u g h t t h a t m u l t i p l i c a t i o n o f h o s t cells was n o t n e c e s s a r y f o r r i c k e t t s i a l g r o w t h . T h e increase in t h e p e r c e n t a g e o f i n f e c t e d cells w h e n i n f e c t e d b l o o d was m i x e d w i t h b l o o d f r o m s u s c e p t i b l e sheep suggested t h a t n e w cells :had b e e n invaded. O n t h e o t h e r h a n d , w h e n p l a s m a f r o m i n f e c t e d s h e e p was m i x e d w i t h b l o o d f r o m s u s c e p t i b l e s h e e p no i n f e c t e d cells w e r e o b s e r v e d a f t e r culture. T h e l a t t e r finding suggests t h a t e i t h e r t h e cell-free f o r m was t o o fragile t o initiate i n f e c t i o n in v i t r o or t h e increases in t h e n u m b e r s o f i n f e c t e d cells w e r e n o t d u e t o n e w cells being i n v a d e d b u t d u e t o t h e c o n t i n u e d m u l t i p l i c a t i o n o f t h e o r g a n i s m s in a l r e a d y i n f e c t e d cells. T h e t e c h n i q u e d e s c r i b e d h e r e p r o v i d e s a m e a n s f o r growin~ t h e o r g a n i s m in s u f f i c i e n t q u a n t i t i e s f o r d e t a i l e d studies o f its p h y s i c a l a n d c h e m i c a l p r o p e r ties a n d f o r i m p r o v i n g antigen yields f o r serological a n d i m m u n o l o g i c a l studies. T h e ability o f the o r g a n i s m t o c o n t i n u e t o p r o p a g a t e in vitro f o r at least 24 h gives a h i t h e r t o i m p o s s i b l e p r o c e d u r e f o r s t u d y i n g t h e m o r p h o l o g i c a l c h a n g e s t h a t o c c u r w i t h i n the i n f e c t e d cell. P r e l i m i n a r y in vitro studies in this d i r e c t i o n indicate t h a t t h e r e is a s e q u e n c e o f d e v e l o p m e n t f r o m single discrete particles to clusters o f particles. ACKNOWLEDGEMENT T h e w o r k was financially s u p p o r t e d , in p a r t , b y t h e Overseas D e v e l o p m e n t Administration, London.
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