parva subsp. parva and T. parva subsp. lawrenceit - Infection and ...

Vol. 53, No. 1

INFECTION AND IMMUNITY, July 1986, p. 186-191

0019-9567/86/070186-06$02.00/0 Copyright © 1986, American Society for Microbiology

Comparative Analysis of Infection and Transformation of Lymphocytes from African Buffalo and Boran Cattle with Theileria parva subsp. parva and T. parva subsp. lawrenceit C. L. BALDWIN,'* M. N. MALU,2 S. W. KINUTHIA,' P. A. CONRAD,' AND J. G. GROOTENHUIS2 International Laboratory for Research on Animal Diseases, Nairobi,' and Wildlife Diseases Section, Kenya Veterinary Research Laboratories, Kabete,2 Kenya Received 13 February 1986/Accepted 1 April 1986

This study compared infection and transformation of peripheral blood mononuclear cells (PBM) of Boran cattle and African buffalo in vitro to determine whether differences occurred which could account for the greater susceptibility of Boran cattle to infection with Theileria parva subsp. parva and T. parva subsp. lawrencei. PBM from buffalo and cattle had a similar percentage of cells which bound T. parva subsp. parva sporozoites (24 to 34%) and in which schizonts developed during the first week after infection (18 to 23%). Using a limiting dilution culture system, it was established, however, that a significantly higher proportion of cattle PBM transformed into continuously replicating cell lines after infection with T. parva subsp. parva than did buffalo PBM. The evidence suggests that the low capacity of T. parva subsp. parva to establish infections in buffalo compared with cattle is related to the lower frequency of buffalo cells which undergo transformation. With T. parva subsp. lawrencei, however, the frequency of transformation of buffalo PBM was higher than that for cattle PBM. The frequency of cells transformed by T. parva subsp. lawrencei, therefore, cannot account for the greater resistance of buffalo to infections with T. parva subsp. Iawrencei. Buffalo must have other mechanisms, either innate or acquired, which control infection with T. parva subsp. lawrencei more efficiently than in cattle.

theilerial sporozoites, the early development of schizonts, and the frequency of precursor cells that can be transformed into continuously replicating cell lines.

Theileria parva is a tick-transmitted protozoan parasite prevalent in East Africa which infects cattle and buffalo. T. parva parasites have been classified into two major subspecies, T. parva subsp. parva and T. parva subsp. lawrencei (23), within which an unknown number of strains exist. Theilerial sporozoites infect lymphocytes of cattle and buffalo and develop into schizonts. This can result in acute lymphoproliferation during which the parasite and host cell divide synchronously (14). Lymphocytes can also be infected with theilerial sporozoites in vitro and may establish continuously replicating cell lines (6). The mechanism by which the parasite transforms the lymphocyte into a continuously replicating cell is unknown. Cattle and buffalo have been shown to differ in their susceptibility to infection with T. parva subsp. parva and T. parva subsp. lawrencei. Infection of cattle with T. parva subsp. parva or T. parva subsp. lawrencei generally results in fatal disease (2, 5, 18). In contrast, buffalo are largely refractory to infection with T. parva subsp. parva (1, 16). Buffalo are also better able to control infection with T. parva subsp. lawrencei than are cattle (4) and invariably become carriers of the infection (25). The present study was undertaken to determine whether differences in the early stages of infection of buffalo and cattle cells in vitro reflect relative susceptibilities of cattle and buffalo to infection in vivo. The susceptibilities of buffalo and cattle peripheral blood mononuclear cells (PBM) to infection in vitro with T. parva subsp. parva and T. parva subsp. lawrencei sporozoites were compared. The parameters of infection evaluated included the capacity to bind

MATERIALS AND METHODS Infection of ticks and isolation of sporozoites. Rhipicephalus appendiculatus ticks were infected with T. parva subsp. parva (Muguga) (5) by feeding nymphal ticks on cattle infected with a tick-derived stabilate (9). R. appendiculatus ticks were infected with T. parva subsp. lawrencei by feeding nymphal ticks on buffalo 5641 (reference date, November 1984), which had been previously infected with sporozoites obtained from ticks fed on carrier buffalo KB2 (22, 24). The carrier status of buffalo 5641 was confirmed by the frequent establishment of Theileria-infected cell lines from the peripheral blood (J. G. Grootenhuis, A. S. Young, T. T. Dolan, B. L. Leitch, D. A. Stagg, and M. N. Malu, manuscript in preparation). After molting, the adult ticks were fed on rabbits for 4 days to stimulate sporozoite development. Tick salivary glands containing parasitized acini were removed under aseptic conditions (8), suspended in sterile unsupplemented RPMI 1640 culture medium (GIBCO Laboratories, Paisley, Scotland), and stored on ice until use. Salivary glands were ground in a glass tissue grinder and centrifuged for 5 min at 100 x g. The supernatant containing sporozoites was collected and used for infection of cells. The infection rate of ticks was estimated by determining the number of infected acini per tick in a sample of 50 female and 50 male ticks, by staining dissected salivary glands (3). The relative concentration of sporozoites in the salivary gland suspensions was estimated from the number of infected acini per tick and is expressed as the number of infected acini suspended per milliliter of medium. A mature infected acinus contains

Corresponding author. t Publication no. 440 of the International Laboratory for Research on Animal Diseases. *

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between 6 x 108 and 109 sporozoites (12). The presence of sporozoites in the supernatant was confirmed by microscopic examination of cytospin smears (Shandon Southern Products Ltd., Cheshire, England) which were fixed in methanol and stained with Giemsa stain. Experimental animals. Cattle used in these studies were uninfected Boran steers (Bos indicus) between 1 and 4 years of age which were reared and maintained at the International Laboratory for Research on Animal Diseases under tick-free conditions. African buffalo (Syncerus caffer), born and maintained at the Wildlife Diseases Section of the Kenya Veterinary Research Laboratories, Kabete, were used between 1 and 2 years of age. They had not been experimentally exposed to T. parva nor did they show any indication of natural infection at the time of the experiments. Theileriafree status was confirmed by examining serum for antibody to Theileria schizonts and piroplasms (7), examining ticks fed on the buffalo for infected salivary gland acini, examining blood smears for piroplasms, and attempting to isolate Theileria-infected cell lines by culturing PBM in vitro (21). Preparation of PBM. Blood taken from buffalo and cattle by jugular venopuncture was collected into an equal volume of Alsever solution. PBM were isolated from cattle blood by using Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, N.J.) gradients as described elsewhere (P. A. Lalor, W. I. Morrison, B. M. Goddeeris, R. J. Jack, and S. J. Black, Vet. Immunol. Immunopathol., in press). Based on trials with buffalo blood with Ficoll-Hypaque solutions of various specific gravities and centrifugation with a variety of speeds and times, the best results for isolation of buffalo PBM were obtained by using Ficoll-Hypaque of specific gravity 1.083 (Histopaque 1083; Sigma Chemical Co., St. Louis, Mo.) and centrifugation at 900 x g for 25 min. Buffalo PBM collected from the gradient were diluted in Alsever solution and pelleted by centrifugation at 800 x g for 10 min. The pelleted cells were washed three times by resuspension in Alsever solution and pelleting at 500 x g for 10 min. Buffalo and cattle PBM were suspended at 107/ml in unsupplemented RPMI 1640 medium for incubation with sporozoites. Binding of sporozoites to PBM. Supernatants from homog-

enized tick salivary glands infected with T. parva subsp. parva (Muguga) were serially diluted with RPMI 1640 medium to concentrations ranging from 16,000 to 2,000 infected acini per ml. Supernatants containing sporozoites were mixed with equal volumes of PBM, vortexed at high speed for 30 s to break up clumps of sporozoites, and incubated at 37°C for 2 h with continuous rotation. This method gave maximal binding of sporozoites to PBM (S. W. Kinuthia, unpublished data). After incubation, PBM were either diluted in complete culture medium for in vitro cultivation as described below or fixed to assess sporozoite binding. For fixation, the PBM and sporozoite mixture was diluted 25-fold with phosphate-buffered saline, and paraformaldehyde was added to a final concentration of 2%. The mixture was incubated at room temperature for 10 min. The fixative was then neutralized by the addition of an equal volume of 50 mM NH4Cl, after which cells were washed twice with phosphate-buffered saline containing 0.2% gelatin. To quantify sporozoite binding, we stained sporozoites with fluorescein-labeled monoclonal antibody (MAb) Dl, which specifically reacts with Theileria sporozoites (11). The percentage of PBM-binding sporozoites and the number of sporozoites bound per cell were assessed by fluorescence microscopy. Two hundred cells were examined per sample. Assessment of schizont development within PBM. PBM were incubated with T. parva subsp. parva (Muguga) sporo-

INFECTION OF LYMPHOCYTES WITH T. PARVA

187

zoites at a ratio of 400 infected acini per 106 cells in 200 RI of RPMI 1640 medium as described above. After incubation with sporozoites, PBM were cultured and monitored for schizont development as described elsewhere (P. A. Lalor, Ph.D. thesis, Brunel University, Uxbridge, England, 1983). Briefly, PBM were diluted with medium, pelleted by centrifugation at 200 x g for 10 min, and suspended to 5 x 105 PBM per ml in complete culture medium consisting of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Hy-Clone Laboratories, Logan, Utah), 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, 5 x 10-5 M 2-mercaptoethanol, 2 mM glutamine, and gentamicin (50 ,ug/ml). PBM were dispensed in 2-ml aliquots into wells of 24-well plates (Costar, Cambridge, Mass.) containing BT6 fibroblasts derived from bovine thymus as feeder cells (15). Twice weekly, one half of the culture medium was replaced with fresh culture medium. Every second day the cells were resuspended in the wells by aspiration with a Pasteur pipette, a 100-,u sample was removed, and the concentration of cells was determined by counting in a hemacytometer. A cytospin smear was prepared to examine cells by light microscopy for schizonts. Cytospin smears were fixed in methanol, acid hydrolyzed as described by Kurtti et al. (15), and stained with Giemsa stain. Assessment of frequency of cells giving rise to transformed cell lines. PBM were incubated with T. parva subsp. parva (Muguga) or T. parva subsp. Iawrencei sporozoites at a ratio of 400 infected acini per 106 PBM in 200 Rl of RPMI 1640 medium as described above for sporozoite binding. After incubation, PBM were pelleted by centrifugation at 200 x g for 10 min and suspended in complete culture medium at 3 x 104 cells per ml. Limiting dilution cultures were set up by the method of Oi and Herzenberg (19) as adapted by Lalor et al. (1Sa). PBM were serially diluted with complete culture medium down to 102 cells per ml. Samples (100 RI) of cells were dispensed into the wells of flat-bottomed 96-well microtiter plates (Costar) containing fibroblasts as feeder cells. Control cultures of PBM not incubated with sporozoites were set up in an identical fashion. Twice weekly, 100 RI of culture medium in each well was replaced with fresh culture medium. Several types of fibroblastic feeder cells were examined for their ability to support growth. Buffalo fibroblastic cells derived from embryonic spleen or peripheral blood were tested for their ability to support the growth of autologous and allogeneic infected buffalo PBM as well as infected cattle PBM. Similarly, bovine fibroblasts were tested as feeder layers for infected buffalo and cattle PBM. No bias according to species or autologous versus allogeneic cells was found. Bovine BT6 cells were therefore used in all tests reported. Twice weekly, wells were examined with an inverted phase-contrast microscope for evidence of cell proliferation. Theileria-transformed cultures were identified by an increase in cell size, formation of cell clumps, and an increase in cell concentration in the wells. In addition, cytospin smears from samples of selected wells were treated by acid hydrolysis, stained with Giemsa stain, and examined for intracellular schizonts. The cultures were monitored for 4 to 5 weeks. The frequency of precursor cells giving rise to transformed cells was assessed by the maximum likelihood solution described by De St. Groth (10) which analyzes the percentage of positive wells over a range of cell concentrations. A portion of the infected cells was propagated in a 24-well plate as described above for assessment of schizont devel-

188

BALDWIN ET AL.

INFECT. IMMUN.

opment, and the resultant infected cells were used for determining the antigenic profile of the schizonts. Some cultures derived from seeding concentrations which gave rise to 30% or less positive wells were further propagated and assessed for their antigenic profile with MAbs reactive with schizonts. These are subsequently referred to as clones although a small proportion may have been derived from more than one cell (13). Antigenic profile of schizonts determined by staining with MAbs. Smears of Theileria-infected cells from uncloned cultures and from selected clones were prepared and screened for reactivity with antischizont MAbs which detect antigenic differences among various T. parva isolates (17). The MAbs used to assess the phenotype of schizonts in the Theileria-infected cells were: MAbs 2 and 3, which were raised by Pinder and Hewett (20) against cattle lymphocytes infected with T. parva subsp. parva (Muguga), and MAbs 17 through 20, which were raised by Newson et al. (18a) against cattle lymphocytes infected with T. parva subsp. lawvrencei.

TABLE 2. Development of T. pari'a subsp. parva (Muguga) schizonts in cattle and buffalo PBM during the first week after infection % PBM containing schizonts at Source of PBM (species [animal no.])

(days postinfection): 3

5

7

Bovine (C210) Bovine (B427) Bovine (B166)

0 0 0

17 23 ND"

17 20 31

Buffalo (6017) Buffalo (6020) Buffalo (6807) Buffalo (6797) Buffalo (6818)

0 0 0 0 0

ND ND 12 17 31

9 5 18 25 34

Bovine (mean + SD)h

0

20.0 ± 4.2

22.7 ± 7.9

Buffalo (mean ± SD)'

0

20.0 ± 9.9

18.2 ± 11.8

ND, Not done. Mean ± standard deviation are for the results indicated in the table for that species. a

RESULTS Percentage of lymphocytes which bound sporozoites. T. parva subsp. parva (Muguga) sporozoites were incubated with PBM from cattle and buffalo to compare the percentage of lymphocytes which had receptors for sporozoites. Table 1 indicates the percentage of lymphocytes that bound one or more sporozoites over the range of sporozoite concentrations tested. A similar percentage of buffalo and cattle PBM bound sporozoites when homogenates of infected acini ranging from 4,000 to 16,000/ml were incubated with PBM. This suggests that the concentration of sporozoites contained within these suspensions was high enough to result in saturation of sporozoite receptors. At the lowest concentration tested (homogenates of 2,000 infected acini per ml), buffalo PBM showed a greater ability to bind sporozoites than did cattle PBM, suggesting either a higher affinity or a greater density of receptors for sporozoites on buffalo cells. Development of schizonts within PBM. The second parameter of infection measured was development of T. parva subsp. parva (Muguga) schizonts. PBM and sporozoites were reacted under conditions that gave maximal binding of sporozoites (4,000 infected acini per ml). Table 2 shows the percentage of lymphocytes which had observable schizonts on days 3, 5, and 7 after infection with sporozoites. Before day 5 no schizonts were detectable in PBM from either cattle or buffalo. The number of cells developing schizonts was the same for the two species at days 5 and 7. Determination of cell numbers in the cultures on days 5 and 7 showed that the TABLE 1. Percentage of buffalo and cattle PBM with one or T. parva subsp. parva (Muguga) sporozoites bound over a range of sporozoite concentrations"

more

%

Source of PBM (Species [animal no.])

Buffalo (6020) Buffalo (6017) Bovine (B166) Bovine (B661)

PBM with

one or more

sporozoites with

the following no. of infected acini/mI of

mediumb: 2,000

4,000

8,000

16,000

33 24 9 9

27 33 24 24

34 32 26 26

30 24 30

22

Results for a representative experiment are shown. b Number of infected acini homogenized per milliliter of medium to yield the sporozoite suspension. Sporozoites at the various concentrations were incubated with a constant concentration of PBM. a

I

concentration of PBM had not detectably altered from the original seeding density. Thus, the percentage of lymphocytes determined to have schizonts at this time is believed to be a reasonable measure of the percentage of PBM in which schizonts initially developed. Transformation of PBM with T. parva subsp. parva (Muguga). PBM from cattle and buffalo were cultured by limiting dilution after incubation with sporozoites (4,000 infected acini per ml) to estimate the proportion of PBM susceptible to transformation after infection. J3uffalo PBM had a much lower frequency of cell line establishment when infected with T. parva subsp. parva (Muguga) than did cattle PBM (Fig. 1; Table 3). The frequency of buffalo PBM transformed was 1.4 to 3.9 logl0 lower than that of cattle PBM. Some clones were selected and propagated further. Over a period of several months, cultures of cattle cells infected with T. parva subsp. parva (Muguga) readily established continuously proliferating cell lines, whereas the majority of buffalo cultures and clones (10 of 13) infected with T. parva subsp. parva (Muguga) did not survive for more than 2 months. No growth was found in control cultures in which PBM not exposed to sporozoites were cultured in bulk or by limiting dilution (data not shown). Transformation of PBM with T. parva subsp. lawrencei. An identical experiment was performed with T. parva subsp. Iawrencei sporozoites to determine whether a similar difference occurred in the frequency of transformation of buffalo and cattle PBM as was found with T. parva subsp. parva (Muguga). Approximately 1 to 2 logl0 fewer cattle PBM established cell lines than buffalo PBM (Fig. 2; Table 3). The frequency of buffalo PBM giving rise to cell lines was 2 to 3 logl0 higher when infected with T. parva subsp. lawrencei compared with that obtained with T. parva subsp. parva (Muguga) (Table 3). Cattle, however, showed the reverse situation. The frequency of cell line establishment was lower after infection with T. parva subsp. lawrencei than with T. parva subsp. parva (Muguga) (Table 3). All buffalo and cattle cultures and clones infected with T. parva subsp. Iawrencei were easily propagated (36 of 36). Antigen phenotype of schizonts in cattle and buffalo PBM.

VOL. 53, 1986


100

-

100

75

-

75

(a2

189

-

0)

.15

0)

50

3._

-

50

C')

:_I

0)

0

0~CL

a.

25

2:]

a

/

1.0

1.5

20

1.0

2.0

1.5

2.5

3.0

3.5

FIG. 1. Limiting dilution cultures of buffalo and cattle PBM infected with T. parva subsp. parva (Muguga) sporozoites. The percentage of positive wells at 4 to 5 weeks after infection for a range of initial cell seeding densities are shown. Results for individual animals are as follows: cattle are open symbols (B166, A; C210, 0; and B427, O), and buffalo are closed symbols (6020, x; 6797, 0; 6807, *; and 6818, A). All cultures were initiated at the same time except those for B166 and 6020 which were initiated together on another occasion.

The antigenic profile of the schizonts was assessed with antischizont MAbs to determine whether schizonts of infected cells reflected the parasite subspecies used for the in vitro infections. This was done to ensure that the growth of infected cells resulted from infection in vitro and did not reflect growth of cells infected previously in vivo. The results with infected cells established in bulk cultures (Table 4) showed that the transformed cells express some or all of the antigens characteristic of the infecting stock of parasite. The antigenic profiles for the clones tested were identical to those of the bulk cultures (data not shown).

FIG. 2. Same as Fig. 1 except that PBM were infected with T. subsp. lawrencei sporozoites. Symbols used are as described in the legend to Fig. 1. All cultures were initiated at the same time.

parva

DISCUSSION

The results of this study suggest that the difference in occurrence of infection of buffalo and cattle with T. parva subsp. parva is not related to the proportion of lymphocytes that are able to bind sporozoites or which permit schizont

development. It is, however, likely to be at least partially attributable to the inefficiency of T. parva subsp. parva to transform buffalo lymphocytes. The frequency of buffalo PBM transformed with T. parva subsp. parva (Muguga) in vitro was considerably lower (1.4 to 3.9 log1o) than for cattle TABLE 4. Antigenic profile of schizonts in cattle and buffalo PBM infected in vitro with T. parva subsp. parva (Muguga) or T. parva subsp. lawrenceia

TABLE 3. Reciprocal of the frequency (1/t) of cells in PBM transformed by T. parva subsp. parva (Muguga) or T. parva subsp. lawrencei sporozoitesa Parasite strain used to infect PBM subsp. parva (Muguga)

parva

1/f

Bovine (C210) Bovine (B427) Bovine (B166)

10 89 4

(6020) (6797) (6807) (6818)

5,140 26,322 2,798 2,196

Buffalo Buffalo Buffalo Buffalo

T.

parva

subsp.

lawrencei

Range of 1/f

1If

Range of 1/f

8-13 70-114 2-5

131 1108 NDb

103-167 874-1407 ND

3,312-7,979 12,546-55,230 1,252-6,252 1,671-2,888

ND 21 23

ND 17-28 18-30

ND

ND

Precursor frequency and range were determined by the maximum likelihood solution described by De St. Groth (10); I/f is the reciprocal of the precursor frequency of cells transformed. b ND, Not done. a

MAbs raised againsta:

Infected cells Parasite strain used to infectb

T.

3.5

3.0

2.5

Log cells / well

Log cells / well

Source of PBM (species [animal no.])

2.0

Source of infected PBM

T. parva subsp. parva (Muguga)

T. parva subsp. lawrencei

2

3

17

18

19

20

Tpp Tpp Tpp

Bovine B427 Bovine C210 Buffalo 6818

+ + +

+ + +

-

-

-

P P P

Tpl Tpl Tpl Tpl Tpl Tpl

Buffalo 5641 Bovine B427 Bovine C210 Buffalo 6807 Buffalo 6797 Buffalo 6818

-

-

+ + + + +

+ + +

-

-

+ + + + + +

+ + + + + +

-

-

+ -

a +, Indicates all schizonts stained at a MAb dilution of 1:400 or greater, except for MAb 17 which stained at a dilution of 1:50; -, indicates a negative reaction at a MAb dilution of 1:50. P, partial, less than 10% of the cells stained at a MAb dilution of 1:200. b Tpp indicates T. parva subsp. parva (Muguga); Tpl indicates T. parva

subsp. lawtrencei (buffalo 5641). c Ticks fed on buffalo 5641 were the source of T. parva subsp. lawrencei sporozoites. The infected cells indicated were isolated from the peripheral blood of buffalo 5641 by in vitro culture.

190

BALDWIN ET AL.

PBM. Furthermore, even after transformation, the growth of buffalo cells was slower, and the long-term survival rate lower, than for cell lines and clones established from infected cattle PBM. The latter observation confirms previous findings (22). It is possible that a T. parv,a subsp. parva (Muguga) transforming agent has a lower affinity for its target in buffalo than in cattle lymphocytes. Alternatively, the transformation frequency may reflect either a rare buffalo lymphocyte type susceptible to transformation or a rare sporozoite type represented in the T. parva subsp. parva (Muguga) stock that is capable of transforming buffalo PBM. The low frequency of transformation of buffalo PBM by T. parva subsp. parva (Muguga) might also have been due to inhibition by other cell types in the cultures. Such an effect would be expected to have a greater influence on the establishment of cell lines at higher cell concentrations. However, limiting dilution cultures did not show a higher relative proportion of transformed cultures as the cell number per well decreased, suggesting that there was no restraint of cell line establishment at high cell concentrations. Furthermore, bulk cultures of buffalo PBM depleted of monocytes did not transform any more efficiently than cultures with between 10 and 20% monocytes present (C. L. Baldwin, unpublished data), indicating that monocytes are not responsible for restraining growth of potentially transformed cells. In contrast to the results obtained with T. parva subsp. parva, in vitro transformation studies with T. parva subsp. Iawrencei indicated that buffalo PBM are more susceptible to transformation than cattle PBM. These results concur with in vivo studies describing the higher virulence of T. parva subsp. lawrencei compared with T. parva subsp. parva in buffalo (1, 4). These results do not explain why buffalo nevertheless are not usually clinically affected by T. parva subsp. Iawrencei while infection of cattle is usually lethal (2, 4, 25). African buffalo may have innate factors which retard the in vivo growth of T. parva subsp. lawrencei-infected cells or they may control infected cells by highly efficient cell-mediated immune responses. These possibilities form the basis of ongoing investigations. The respective abilities of T. parva subsp. parva and T. parva subsp. lawrencei to transform cattle and buffalo PBM may provide another means to distinguish the two subspecies of T. parva in vitro in addition to the MAb characterization of schizont antigens. Additional stocks of the two subspecies need to be evaluated to confirm that this is a valid parameter. The difference in ability of these two stocks to transform buffalo and cattle PBM may also provide a useful experimental system in which to study mechanisms of transformation, including possible polymorphic parasite products which induce transformation, as well as the host-cell target of these products. ACKNOWLEDGMENTS We would like to thank P. Belsoi, R. Njamunggei, and B. Sohanpal for excellent technical assistance; B. Leitch, D. Stagg, and W. Voigt for providing sporozoites; J. Newson for MAbs against T. parva subsp. lawrencei schizonts; J. Young for writing the computer program for determining the precursor frequency of transformed lymphocytes; and S. Black for suggestions on experimental approach and helpful discussions. LITERATURE CITED 1. Barnett, S. F., and D. W. Brocklesby. 1966. The susceptibility of the African buffalo (Syncerus caffer) to infection with Theileria

INFECT. IMMUN.

parva (Theiler, 1904). Br. Vet. J. 122:379-386. 2. Barnett, S. F., and D. W. Brocklesby. 1966. The passage of Theileria Iawrencei (Kenya) through cattle. Br. Vet. J. 122:396-409. 3. Blewett, D. A., and D. Branagan. 1973. The demonstration of Theileria pari'a infection in Rhipicephalus appendiculatus salivary glands. Trop. Anim. Health Prod. 5:27-34. 4. Brocklesby, D. W., and S. F. Barnett. 1966. The isolation of "Theileria lIavrencei (Kenya)" from a wild buffalo (Svnceruis caffer) and its serial passage through captive buffaloes. Br. Vet. J. 122:387-395. 5. Brocklesby, D. W., S. F. Barnett, and G. R. Scott. 1961. Morbidity and mortality rates in East Coast fever (Thleileria pari'a infection) and their application to drug screening procedures. Br. Vet. J. 117:529-531. 6. Brown, C. G. D., D. A. Stagg, R. E. Purnell, G. K. Kanhai, and R. C. Payne. 1973. Infection and transformation of bovine lymphoid cells in vitro by infective particles of Theileria parla. Nature (London) 245:101-103. 7. Burridge, M. J., A. S. Young, D. A. Stagg, G. K. Kanhai, and C. D. Kimber. 1974. Theileria lawrencei infection of cattle and African buffalo: evaluation of a buffalo cell culture schizont antigen for the indirect fluorescent antibody test. Res. Vet. Sci. 17:285-289. 8. Buscher, G., W. I. Morrison, and R. T. Nelson. 1984. Titration in cattle of infectivity and immunogenicity of autologous cell lines infected with Theileria parva. Vet. Parasitol. 15:29-38. 9. Cunningham, M. P., C. G. D. Brown, M. J. Burridge, A. D. Irvin, R. E. Purnell, and D. E. Radley. 1973. Theileria parva: comparative infectivity of a ground tick stabilate and a classical 10-tick challenge. Res. Vet. Sci. 15:263-265. 10. De St. Groth, S. J. 1982. The evaluation of limiting dilution assays. J. Immunol. Methods 49:R11-R23. 11. Dobbelaere, D. A. E., P. R. Spooner, W. C. Barry, and A. D. Irvin. 1984. Monoclonal antibody neutralizes the sporozoite stage of different Theileria parl'a stocks. Parasite Immunol. 6:361-370. 12. Fawcett, D. W., A. S. Young, and B. B. Leitch. 1985. Sporogeny in Theileria (Apicomplexa Piroplasmida). A comparative ultrastructural study. J. Submicrosc. Cytol. 17:299-314. 13. Henry, C., J. Marbrook, D. C. Vann, D. Kodlin, and C. Wofsy. 1980. Limiting dilution analysis, p. 138-152. In B. B. Mishell and S. M. Shiggi (ed.), Selected methods in cellular immunology. W. H. Freeman & Co. Publishers, San Francisco. 14. Hulliger, L., J. K. H. Wilde, C. G. D. Brown, and L. Turner. 1964. Mode of multiplication of Theileria in cultures of bovine lymphocytic cells. Nature (London) 203:728-730. 15. Kurtti, J. F., U. D. Munderloh, A. D. Irvin, and G. Buscher. 1980. Thleileriia parva: early events in the development of bovine lymphoblastoid cell lines persistently infected with macroschizonts. Exp. Parasitol. 52:280-290. 15a.Lalor, P. A., W. I. Morrison, and S. J. Black. 1986. Monoclonal antibodies to bovine leukocytes define heterogeneity of target cells for in v,itro parasitosis by Theileria parva, p. 72-87. In W. I. Morrison (ed.), The ruminant system in health and disease. Cambridge University Press, Cambridge, England. 16. Lewis, E. A. 1943. East Coast fever and the African buffalo, the eland and the bushbuck. East Afr. Agric. J. 9:90-92. 17. Minami, T., P. R. Spooner, A. D. Irvin, J. G. R. Ocama, D. A. E. Dobbelaere, and T. Fujinaga. 1983. Characterization of stocks of Theileria parva by monoclonal antibody profiles. Res. Vet. Sci. 35:334-340. 18. Neitz, W. 0. 1955. Corridor disease: a fatal form of bovine theileriosis encountered in Zululand. Bull. Epizoot. Dis. Afr. 3:121-123. 18a.Newson, J., J. Naessens, D. A. Stagg, and S. J. Black. 1986. A cell surface antigen associated with Theileria parv'a lawrenceiinfected bovine lymphoid cells. Parasite Immunol. 8:149-158. 19. Oi, V. T., and L. A. Herzenberg. 1980. Immunoglobulinproducing hybrid cell lines, p. 351-372. In B. B. Mishell and S. M. Shiggi (ed.), Selected methods in cellular immunology. W. H. Freeman & Co. Publishers, San Francisco. 20. Pinder, M., and R. S. Hewett. 1980. Monoclonal antibodies

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detect antigenic diversity in Theileria parva parasites. J. Immunol. 124:1000-1001. 21. Stagg, D. A., C. G. D. Brown, J. G. Crawford, G. K. Kanhai, and A. S. Young. 1974. In vitro cultivation of Theileria lawrencei-infected lymphoblastoid cell lines derived from a buffalo (Syncerus caffer). Res. Vet. Sci. 16:125-127. 22. Stagg, D. A., A. S. Young, B. L. Leitch, J. G. Grootenhuis, and T. T. Dolan. 1983. Infection of mammalian cells with Theileria species. Parasitology 86:243-254. 23. Uilenberg, G. 1981. Theilerial species of domestic livestock, p. 4-37. In A. D. Irvin, M. P. Cunningham, and A. S. Young (ed.),


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