Titration in cattle of infectivity and immunogenicity of autologous cell lines infected with Theileria parva

Veterinary Parasitology, 15 (1984) 29--38 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

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TITRATION IN CATTLE OF INFECTIVITY AND IMMUNOGENICITY OF AUTOLOGOUS CELL LINES INFECTED WITH THEILERIA PAR VA

G. B~/SCHER*, W.I. MORRISON and R.T. NELSON

International Laboratory for Research on Animal Diseases (ILRAD), P 0 Box 30709, Nalrobl (Kenya) (Accepted for publication 25 October 1983)

ABSTRACT Buscher, G., Morrison, W.I. and Nelson, R.T., 1984. Titration in cattle of infectivity and immunogenicity of autologous cell lines infected with Theilerla parva. Vet Parasltol., 15 29--38. Cell lines infected with Thederla parva were derived by infection of bovine peripheral blood lymphocytes with sporozoites in vitro. Cattle were inoculated with doses of autologous infected cells ranging from l x 101 to l x 108 . Infection became established in animals which received l x 102 or more cells. While l x 102 cells resulted in sub-patent infection with development of immunity to challenge with sporozoites, larger doses of cells gave rise to patent infections of increasing severity. Thus, doses of lX 10 s and l x 106 cells sometimes produced lethal infections and with l x 107 and l x 108 the outcome was invariably lethal. Based on the previous observation that induction of immunity by allogeneic cells requires transfer of infection into the recipient-host cells, a comparison of the infections produced by autologous and allogeneic cells indicated that the transfer of infection from allogeneic cells occurs at a frequency of maximally l z 10 -5 . Two pairs of cattle were identified as being mutually non-reactive in the mixed leukocyte reaction (MLR). Doses of l x 106 and l x 107 cells of cell lines derived from 1 animal of each pair were inoculated into the autologous host, the non-reactive partner and an animal which was shown to be strongly reactive to the donor in the MLR. In each mstance, the reaction in the MLR non-reactive recipient was not significantly different from that of the MLR reactive recipient, but was markedly different from that of the autologous recipient.

INTRODUCTION East Coast Fever (ECF), an economically important disease of cattle in E a s t a n d C e n t r a l A f r i c a , is c a u s e d b y t h e i n t r a - c e l l u l a r p r o t o z o a n p a r a s i t e Thederia p a r v a , w h i c h is n a t u r a l l y t r a n s m i t t e d b y t h e t i c k R h i p i c e p h a l u s appendiculatus. F o r e x p e r i m e n t a l studies of the disease 3 m e t h o d s of initiating i n f e c t i o n in cattle have b e e n used: a p p l i c a t i o n of i n f e c t e d ticks, i n o c u l a t i o n *Present address: Institut fur Parasitologie, Bunteweg 17, D-3000 Hannover 71, W. Germany. 0304-4017/84/$03.00

© 1984 Elsevier Science Publishers B.V.

30 of suspensions of infective sporozoites derived from ticks (Cunningham et al., 1973a) and inoculation of allogeneic bovine lymphoblasts infected with macroschlzonts, the pathogenic stage in cattle (Wilde, 1967). The variation in the reactions of cattle inoculated with the same dose by any of these 3 methods can be explained by differences in susceptibility of individual cattle or by a lack of u m f o r m i t y of the inocula. The number of parasites inoculated during tick challenge can vary greatly from one group of ticks to another (Purnell et al., 1974) and, therefore, result m different courses of infection (Brocklesby et al., 1961). The variability of bovine reactions after inoculation of sporozoite suspensions (Cunningham et al., 1974) is commonly t h o u g h t to be caused by "clumping" of the sporozoites (Wilde, 1967). That cattle react differently to the same dose of infected heterologous cells, may be due to variation in the degree of histocompatibitity differences between the recipient and the donor cells, thus allowing a different number of schlzonts to be transferred into the recipmnt's cells. Thin transfer is considered essential for infecting the recipient (Brown et al., 1978; Emery et al., 1981b). To improve the predmtabfllty of the course of refection with respect to the moculum, we studmd the refection in cattle inoculated with graded numbers of autologous cells transformed in vitro by infection with sporozoites of T. parva. Of partmular interest was the dose-level required to estabhsh refection, and the dose-level which invariably resulted in lethal infection. If the difference between these dose-levels was large and different doses within this range resulted in distinctly different courses of infection, a more accurate m e t h o d of challenge would be avmlable for studying the dmease. It would also be possible to dissect the immune response against the schizont w i t h o u t confusion with responses against sporozoltes (Gray and Brown, 1981; Musoke et al., 1981) or major histocompatibility antigens following the inoculation of infected allogenic cells. MATERIALS AND METHODS Cattle

All of the cattle used, except 2, were Bos taurus, either Friesian or Hereford crosses. The remaining 2 animals (Nos. 18 and 19) were Borans (B. mdicus). All animals were purchased as weaners from Kenyan farms known to be free from ECF. At ILRAD they were fed on a concentrated ration supplemented with hay. At the time of the experiments they were 12--18 months old and weighed 200--300 kg. All animals had been consistently negative in m o n t h l y immunofluorescence tests for antibodies against T. parva macroschizonts (Goddeeris et al., 1982). Parasite T. parva (Muguga) was used m its third cattle--tick passage after the stock had been obtained as stabilate No. 110 from the East African Veterinary

31 Research Organization. R. appendiculatus (Muguga) treks, also originally from that laboratory, were fed in their nymphal stage on cattle with piroplasmic parasitemia. Nymphs were collected from the cattle and 2 months after moulting the infected adults were pre-fed on rabbits for 5 days. The female ticks were cleaned by immersion in 70% ethanol with occasional stirring for 5 min. After 3 rapid washes in sterile distilled water, they were fixed with insect pins to paraffin wax in a petri dish and immersed in complete culture medium (see below). Their chitin back was removed with imd e c t o m y scissors and fine forceps and the salivary glands taken out and transferred into complete medium on ice. The salivary glands of 40--60 female ticks were ground in an ice-cold glass tissue grinder and centrifuged at 100 g at room temperature for 5 min. The resultant supernatant was used for the in vitro refection.

Cell hnes Cell lines were derived by a modification of the method originally described by Brown et al. (1973). Bovine peripheral blood leukocytes (PBL) were isolated on Ficoll-Paque (Pharmacia, Uppsala, Sweden), as described by Emery and McCullagh (1980), and partially depleted of adherent cells by incubation in a T25 plastic culture flask (Costar, Cambridge, MA, U.S.A.) at 37°C for 1 h. The non-adherent cells were then spun at 6 0 0 g for 10 min at room temperature and resuspended at a concentration of 2.5 × 106 cells ml -~ in Leibovitz 15 medium (Flow Laboratories, Irvine, Scotland) supplemented with 10% tryptose phosphate broth (Oxoid, Basingstoke, England), 20% heat-inactivated foetal bovine serum (Flow Laboratories), 10 gg m1-1 glutamine, 100 iu m1-1 penicilhn and 100 pg m 1-1 streptomycin (complete culture medium). Aliquots of the sporozoite suspension were then added to the leukocytes and the mixtures placed in 24-well (2 ml/well) tissue culture plates (Linbro Scientific Inc., Hamden, CT, U.S.A.) containing a feeder layer of cells derived from foetal bovine thymus (BT6). The cultures were incubated at 37°C in an atmosphere of 5% CO2 in air and the medium was changed every 2--3 days. After 3 weeks, the cells were removed from the plates and placed in T25 flasks containing the same feeder layer. After a further 5--10 days they were transferred to flasks without feeder layers. Only vigorously growing lines were maintained for experiments and at the time of inoculation the maximum cultivation time was 80 days.

Experimental design A group of 24 cattle was screened in a one-way mixed leukocyte reaction (MLR). The M L R was performed using PBL as described previously (Emery and Morrison, 1980). Two pairs of animals (Nos. 10 and 14; 11 and 16) were identified whose PBL were mutually non-reactive in the MLR; on the 4 occasions when the test was performed, the stimulation index between each

32

pair was always less than 1.5. Infected cell lines were derived from 1 animal of each pair (Nos. 10 and 11) and these formed part of the group which were inoculated with autologous infected cells (see below). At the time each of these animals was inoculated with autologous cells, the non-reactive partner (Nos. 14 and 16) plus an animal which showed strong MLR reactivity (Nos. 15 and 17) received the same dose of cells as shown in Table I. Two allogeneic animals (Nos. 18 and 19) were also inoculated with 1× 108 infected cells of the cell line derived from animal No. 13. TABLEI E x p e r i m e n t a l a r r a n g e m e n t o f c a t t l e a c c o r d i n g t o t h e i r M L R r e s p o n s i v e n e s s t o t h e cell d o n o r a n d t h e i r r e s p o n s e s t o i n o c u l a t i o n w i t h Theilema parva-infected cells a n d s u b s e q u e n t s p o r ozoite challenge Ammal

MLR-reaction with donor

Dose of cells

R e s p o n s e t o i n f e c t e d cells Patent refection

Death

Antlmacroschizont antibodies

Response to challenge with sporozoites

10 ( D o n o r ) 14 ( R e c i p i e n t ) 15 ( R e c l p m n t )

Autologous Non-reactor Reactor

106 106 10 ~

+ ---

+ ---

11 ( D o n o r ) 16 ( R e c i p i e n t ) 17 ( R e c i p i e n t )

Autologous Non-reactor Reactor

10" 10 ~ 10 ~

+ + --

+ ---

+ +

Immune Immune

13 ( D o n o r ) 18 ( R e c i p i e n t ) 19 ( R e c i p i e n t )

Autologous Reactor Reactor

10 ~ 109 10 ~

+ + +

+ ---

+ +

Immune Immune

Susceptible Susceptible

Thirteen cattle were inoculated subcutaneously oelow the left ear with different numbers of cultured autologous infected cells ranging from 1× 10' to 1× 108 . All cultured cells were m the log phase of growth when inoculated, and the dose was based on the number of viable infected cells. All animals whmh survived inoculation with infected cells were examined for the presence of anti-macroschlzont antibodies by the indirect immunofluorescence test (Goddeeris et al., 1982). With the exception of animal No. 7, all of these survivors, along with 4 control animals, were challenged 2--3 m o n t h s later with a dose of a T. parva (Muguga) tick-derived stabilate prepared as described by Cunningham et al. (1973b), which in previous ex periments had been lethal for 38 out of 40 cattle inoculated. Momtoring o f infection Infection was detected by examining Giemsa-stained smears of lymph node puncture biopsies (Radley, 1971). Biopsies of the regional lymph node

33 were performed from Day 1 onwards in cattle which received cell lines, and from Day 5 onwards in animals challenged with tick-derived stabilate. Once parasites were detected in the drainage node, sampling was switched the following day to the contralateral lymph node. Rectal temperatures were recorded daffy from Day 1 onwards. Because of the difficulty in accurately quantifying the parasites in cattle (Morrison et al., 1981), several parameters were used to evaluate the course of the infections. (a} Pre-patent days (PPD): this is defined as the number of days between inoculation and the first of at least 2 consecutive days with schizonts microscopically detectable in the regional node. (b) Days to fever (DTF); this is defined as the number of days between inoculation and the first of at least 2 consecutive days of a rectal temperature above 39.5°C. (c) Days to spread (DTS); this is defined as the number of days between inoculation and the appearance of schizonts in any lymph node other than the regional node. (d) Days to death (DTD). (e) Days to recovery (DTR): this is defined as the number of days between inoculation and the first of at least 2 consecutive days on which schizonts could n o t be found in a 5-min search of the biopsy smear from a lymph node, other than the regional node. RESULTS The results of the inoculation of autologous cells are shown in Table II. Doses of 1X 103 or more autologous cells caused patent infections in cattle. The severity of the reaction increased with increasing doses, as shown by shorter time-intervals to the detection of parasites, earlier fever and shorter time to death at the higher doses. The period of patent infection in survivors was about the same at different doses. One out of 2 cattle receiving 1X 10: and 1 out of 2 cattle receiving 1X 106 autologous cells died. Higher doses were invariably lethal. In animal No. 12 schizonts were seen transiently in the local node on Day 1 and then from Day 6 onwards. The 2 animals which had received 1× 102 autologous cells, and 1 out of the 2 cattle which had received l × 101 autologous cells developed an antibody response to T. parva macroschizonts, as detected in the immunofluorescence test, indicating that infection had become established. The other animal stayed serologically negative after the inoculation of 1 X 101 autologous cells. Table III shows the results of the challenge with sporozoites of the homologous T. parva stock, of the cattle surviving the moculation with autologous cells. All cattle surviving a patent infection, or surviving a latent infection with positive serological reaction, proved resistant to a challenge that was lethal for 4 control cattle (Nos. 20--23). Both calves which had received 1X 101 autologous cells had a severe reaction, but only the one which had not reacted serologically died.

34 T A B L E II R e a c t i o n s o f c a t t l e t o i n o c u l a t i o n o f Theiler~a parva-infected a u t o l o g o u s cells Animal

Dose

R e s p o n s e t o a u t o l o g o u s cells Pre-patent days a

I(H)

b

I0'

-- d

--

2(F)

c

I0'

--

--

Days to spread a

Days to death a

Days to recovery a

3(F)

102

--

--

4(S)

10 2

--

--

5(H)

I0 ~

I0

I0

12

15

5 x 10 ~ 10 ~ 10 ~ 106 10 ~ 10 ~ 10 ~ 10 ~

7 6 5 6 4 4 6 4

10 S 6 9 7 6 5 7

10 9 9 10 9 S 7 5

14

6 (H) 7 (F) 8(H) 9(F) 10(H) ll(H) 12(H) 13(F) a b c d

Days to fever a

14

22 12 22 18 19 12

F u l l d e f i n i t i o n s are given in " M a t e r i a l s a n d M e t h o d s " . (H) H e r e f o r d cross. ( F ) F r i e s i a n cross. --, n o r e a c t i o n .

TABLE III R e a c t i o n s o f c a t t l e t o c h a l l e n g e w i t h s p o r o z o i t e s o f t h e h o m o l o g o u s Theilerza parva s t o c k a f t e r s u r v i v i n g t h e i n o c u l a t i o n w i t h a u t o l o g o u s cells Animal

Pre-patent days a

Daysto fever a

Daysto spread a

Daysto death a

1 2

8 10

8 8

11 12

19

3

--C

--

4 5

--

6

--

9 20 21 22 23

5

b b b b

--

18

--

--

9 6 7 7 7

-7 8 8 8

-10 10 10 10

Daysto recovery a

21 17 21 22

a F u l l d e f i n i t i o n s are g i v e n in " M a t e r i a l s a n d M e t h o d s " . b Challenge control. c --, no reaction.

35

As shown in Table I, the 2 cattle (Nos. 14 and 15) which were selected as non-reactive and reactive, respectively, in the M L R to PBL of animal No. 10 both remained microscopically and serologically negative following inoculation of 1X 106 cells of the cell line from animal No. 10. Furthermore, both animals succumbed to the lethal challenge with sporozoites. Of another pair of cattle, similarly selected on the basis of MLR reactivity and inoculated with 1X 107 cells derived from animal No. 11, the non-reactor (No. 16) showed schizonts in the regional node on Day 4 and had several days of elevated b o d y temperature. The other (No. 17) showed no reaction. However, both became positive serologically and were immune to the challenge with sporozoites. Following challenge, the non-reactor showed schizonts in the regional node on Day 9 only, while No. 17 remained negative. Two B. indicus cattle (Nos. 18 and 19), which received the same inoculum (1X 108 cells) as animal No. 13, became transiently positive in the drainage node. These animals did not exhibit a rise in rectal temperature and the infection did not spread to other lymph nodes. Both animals developed an antibody response to T. parva schizonts. After challenge with sporozoites, one did not show any reaction while the other developed a transient infection detectable only in the regional lymph node. DISCUSSION

The present study has shown that, as with tick challenge (Jarrett et al., 1969), sporozoite titration (Cunningham et al., 1974) and inoculation of allogeneic infected cells (Pirie et al., 1970; Cunningham, 1977), the severity of the course of infection following inoculation with autologous cells was related to the number of parasites administered. However, by comparison with allogeneic infected cells, much smaller doses of autologous cells lead to infection and immunity. Apart from the dose levels of 1X 10 s and 1X 106 , distinct differences in the course of infection were observed in animals receiving different doses of cells and the results were fairly consistent for animals at each dose-level. However, at doses of 1X l 0 s and 1X 106 , although the initial course of infection was similar in each pair of animals, one animal survived whereas the other died. Interestingly, in each instance, the animal which died was a Hereford cross and the animal which survived was a Friesian cross. In very limited comparisons of susceptibility of B. taurus breeds to single or graded doses of sporozoites, no difference in susceptibility has been found (G. Bfischer, unpublished results, 1980). Another explanation for the lack of uniformity in reaction to the same inoculum may lie in qualitative differences in the cell lines, a possibility which we hoped to control by using only similarly vigorously growing cells in the log phase after a similar period in culture. The shortest pre-patent period, 4 days, was achieved with doses of 1X 106--1X 10 s cells. Four days is also the minimum pre-patent period observed following inoculation of sporozoites and, as with autologous cells,

36 no shorter pre-patent period is f o u n d even after large doses of sporozoites (Radley et al., 1974). The inability to detect parasites in the first 3 days after inoculation, along with the quick succession of the appearance of schizonts in the regional lymph node and then in other nodes in animals No.12 and 13, suggests that only a proportion of the cells inoculated was immediately retained in the regional node. Thus, only after several days of multiplication could the retained cells be detected. Only in animal No. 12 were the inoculated cells transiently detectable in the draining node on Day 1. There is also evidence, following inoculation of sporozoites, that not all parasites are trapped in the drainage node, as shown by Wilde et al. (1966) and Emery (1981), who could not prevent calves from becoming patently infected after extirpation of the regional lymph node during the pre-patent period. Although inoculation of 1X 105 autologous cells did not result m patent infection, the animals m o u n t e d an a n t i b o d y response to macroschlzonts and were immune to lethal sporozoite challenge. The effect of 1× 101 cells was not clear; whereas animal No. 1 was obviously fully susceptible at the time of sporozoite challenge, animal No. 2 probably was not. This animal might have been a fortuitous survivor, as previously 2 out of 40 calves had survived the same sporozoite dose. On the other hand, the fact that it developed antibodies against T. parva suggests that the 1X 101 autologous cells had established an Infection, as large numbers of parasites are needed to elicit an antibody response (Emery et al., 1982). Recent studies have shown that induction of i m m u n i t y to refection with T. parva correlates with development of a genetically restricted cell-mediated cytotoxic response against autotogous macroschizont-infected cells (Eugui and Emery, 1981; Emery et al., 1981a). This indicates that for induction of i m m u n i t y , ammals may require to be confronted with the parasite m cells of their own genotype and implies that, during immunization with allogeneic cell lines, there must be transfer of infection into cells of the recipient. The findings in the present study, that the minimum numbers of autologous and allogeneic cells which resulted in i m m u n i t y were 1X 102 and 1X 107 , respectively, indicate that the rate of schizont transfer is approximately 1X 10 5. However, the likelihood that allogeneic infected cells continue to proliferate for several days followmg inoculation implies t h a t the frequency of transfer may be considerably less than 1X 10 -s . Although the 2 cattle which received 1X 10 s allogeneic cells developed patent infections, it is unlikely that sufficient numbers of the recipients' cells could have become infected to give such a short pre-patent period and, if they did, the infection would almost certainly have become generalized. Thus, the infected cells which were detected were probably part of the inoculated allogeneic population. The selection of recipient animals whose cells were mutually non-reactive in an MLR With those of the donor made little difference to the course of infection and i m m u n i t y induced by allogeneic infected cells. Thus, homology of the lymphocyte-defined major histocompatibility (MHC) antigens

37 d e t e c t e d in t h e M L R d o e s n o t a p p e a r t o e n h a n c e t h e degree o f t r a n s f e r o f i n f e c t i o n , or t h e c a p a c i t y t o i m m u n i z e w i t h allogeneic i n f e c t e d cells. T h e s e results i n d i c a t e t h a t d i f f e r e n c e s in t h e serologically d e f i n e d d e t e r m i n a n t s o f t h e b o v i n e M H C are s u f f i c i e n t t o e n s u r e r a p i d r e j e c t i o n o f allogeneic Theileria-infected lymphocytes. In c o n c l u s i o n , t h e p r e s e n t s t u d y has s h o w n t h a t it is possible t o i n d u c e lethal i n f e c t i o n s o r s u b l e t h a l i n f e c t i o n s o f v a r y i n g severity b y i n o c u l a t i o n o f c a t t l e w i t h d i f f e r e n t doses o f a u t o l o g o u s cells i n f e c t e d w i t h T. parva. This will p r o v i d e a useful s y s t e m f o r dissecting t h e i m m u n e r e s p o n s e s d i r e c t e d against t h e m a c r o s c h i z o n t - i n f e c t e d cell, as it e x c l u d e s r e s p o n s e s elicited b y s p o r o z o i t e s ( G r a y a n d B r o w n , 1 9 8 1 ; M u s o k e et al., 1 9 8 2 ) a n d avoids possible c o n f u s i o n b y r e s p o n s e s t o M H C antigens f o l l o w i n g i n o c u l a t i o n o f allogeneic m a c r o s c h i z o n t - i n f e c t e d cells. ACKNOWLEDGEMENTS We are g r a t e f u l t o C.G.D. B r o w n ( p r e v i o u s l y at E A V R O ) f o r p r o v i d i n g S t a b i l a t e 1 1 0 ( T h e i l e r i a parva, Muguga) a n d R h i p i c e p h a I u s a p p e n d i c u l a t u s (Muguga), a n d B. O t i m , J. Tangus, P.M. K a r o k l a n d P.K. M b u r u f o r e x c e l l e n t assistance. T h a n k s are also d u e to B. G o d d e e r i s f o r p e r f o r m i n g t h e serological tests a n d C. M u n y u a f o r t y p i n g t h e m a n u s c r i p t . This is I L R A D publication No. 211.

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