Immunization of cattle against Theileria parva bovis and their exposure to natural challenge

Veterinary Parasitology, 37 ( 1 9 9 0 ) 185-196 Elsevier Science Publishers B.V., A m s t e r d a m

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Immunization of cattle against Theileria parva bovis and their exposure to natural challenge H.T. Koch ~'*'*, L. Kambeva l, J.G.R. Ocama ~'*, F.C. Munatswa ~, F.F.J. Franssen 2, G. Uilenberg 2"**, T.T. Dolan 3 and R.A.I. NorvaF '*** Veterinary Research Laboratory, P.O. Box 8101, Causeway, Harare (Zimbabwe) 2Department of Tropical Veterinary Medicine and Protozoology, Faculty of Veterinary Medicine, University of Utrecht, P.O. Box 80.165, 3508 TD Utrecht (The Netherlands) 31nternational Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi (Kenya) (Accepted for publication 6 June 1990)

ABSTRACT Koch, H.T., Kambeva, L., Ocama, J.G.R., Munatswa, F.C., Franssen, F.F.J., Uilenberg, G., Dolan, T.T. and Norval, R.A.I., 1990. Immunization of cattle against Theileria parva bovis and their exposure to natural challenge. Vet. Parasitol., 37:185-196.

Theileria parva boris isolates were tested for their immunizing capacity under natural field challenge on Willsbridge Farm in the highveld of Zimbabwe. Fifteen susceptible Sussex yearlings were immunized with the Boleni stock and 15 with a mixture of three isolates from the farm, using tickderived sporozoite stabilates. No chemoprophylaxis was used. A dose of 0.1 ml of stabilate appeared to be safe in preliminary laboratory experiments, but the reactions were severe in the Sussex cattle and one died despite treatment. Twenty-nine immunized animals and 10 controls first experienced a mild infection, starting about 15 days after their arrival at the farm. Ten of the immunized animals and four controls had schizonts in peripheral lymph nodes for variable periods; one third of those had pyrexia. Nymphal Rhipecephalus appendiculatus ticks applied to three of the reacting immunized calves transmitted Theileria taurotragi to two animals and T. parva to a third. A second Theileria infection, due to T. parva boris, was detected shortly after the first one. Schizonts were detected in seven out of 10 controls. Pyrexia was more severe and prolonged. Two of the controls died of theileriosis. At the same time schizonts were seen in three immune animals and eight of them had short periods of pyrexia. Intercurrent infections with Babesia bigemina, Borrelia theileri and Eperythrozoon were detected and may have contributed to the fever. Tick infestations were low during the exposure. In the second year of exposure, four out of eight new control animals had severe reactions, and one Present addresses: *Redd B a r n a M o z a m b i q u e P r o g r a m m e , P.O. Box 4581, Harare, Zimbabwe. **IEMVT, 10, rue Pierre Curie, 94704 Maisons-Alfort Cedex, France. * * * D e p a r t m e n t of Infectious Diseases, College of Veterinary Medicine, University of Florida, Building 471, Mowry Rd., Gainesville, FL 32611-0633, U.S.A. *FAO staff, assigned to Project G C P / Z I M / 0 0 4 / D E N .

0304-4017/90/$03.50

© 1990 - - Elsevier Science Publishers B.V.

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died. None of the immunized animals became ill, but one animal from the first year control group, which had not reacted previously, had clinical theileriosis. It is concluded that immunization provided an effective protection against field challenge.

INTRODUCTION

Theileria parva is an important tick-transmitted protozoan parasite of cattle, causing East Coast fever in east and central Africa, January disease in Zimbabwe, and Corridor disease in those parts where cattle share grazing with buffalo. This paper follows the trinomial system of nomenclature recomm e n d e d by Uilenberg (1976) and Lawrence (1979), in which the parasite causing January disease is called T. parva boris. January disease is confined to the highveld of Zimbabwe and causes 200-900 deaths per year (Thomson, 1985 ). Outbreaks occur seasonally in the wet period of the year (DecemberMarch ), when the adult stage of the tick vector Rhipicephalus appendiculatus is active. The 1-year life cycle of the vector is determined by climatic conditions affecting the adult stage of the tick ( Short and Norval, 1981 ). About one million cattle were lost after disruption of tick control during civil war in the 1970s (Lawrence et al., 1980). This experience stimulated a new approach to the control of tick-borne diseases based on endemic stability. As intensive use of acaricides had failed to eradicate tick-borne diseases from parts of Zimbabwe, especially the commercial farms, it might be safer and more economic to rely on the immunity of the herd to control these diseases (Lawrence et al., 1980 ). January disease regularly causes mortality even when enzootic. Therefore a vaccine against January disease was needed. Because East Coast fever had been eradicated in Zimbabwe some 35 years ago (Matson, 1967), it was decided that Zimbabwe should develop its vaccine based upon indigenous parasites. This paper describes the first January disease immunization field trial. MATERIALS A N D M E T H O D S

Animals Pure-bred Sussex weaners, about 14 months old, were obtained from a farm without any history of pathogenic theileriosis. Serological titres to Theileria taurotragi (Lawfield stock) (Lawrence and Mackenzie, 1980) piroplasm antigen in the indirect fluorescent antibody test (IFA test) (Burridge, 1971 ) were recorded, but the animals were negative to T. parva boris (Boleni stock), (Lawrence and Mackenzie, 1980) piroplasm antigen. Piroplasms were observed in some animals. Before field exposure, the 30 animals to be immunized were kept in small paddocks at the Veterinary Research Laboratory

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(Harare). The 10 control animals were kept at the Experimental Farm at Mazowe. Both groups were sprayed weekly with amitraz (Triatix, Cooper). The eight control animals for the second year of exposure came from another farm without any history of theileriosis and were negative for antibodies to T. parva antigen. Immunizing stabilates Tick-derived stabilates were prepared from infected R. appendiculatus adults according to the m e t h o d of Cunningham et al. ( 1973 ), at a concentration of 10 ticks ml-1 medium. The Boleni stock had been passaged several times before the immunizing stabilate, 85-4, was made. The tick batch used for this stabilate had 1585 infected acini per 50 ticks. The stabilate dose was chosen based on testing doses of 0.01, 0.1, 1 or 10 ml in 29 cattle. Two animals out of 19 died after infection with 1 ml and all surviving cattle were i m m u n e to subsequent homologous challenge. The Willsbridge isolates 86-2, 86-3 and 86-4 originated from three bait cattle which had become infected on Willsbridge Farm in January 1986. Tick batches fed on them had infection rates of 263, 1218 and 271 infected acini per 50 ticks, respectively. Fourteen cattle had been infected with this stabilate mixture (equal volumes) in previous titration experiments; four with 0.01 ml and 10 with 1 ml. Four of the cattle infected with 1.0 ml had died of theileriosis. Four infected with 0.01 ml and four infected with 1 ml were given a homologous challenge and proved to be immune. Animals immunized with either Boleni or Willsbridge isolates, resisted reciprocal challenge (Koch et al., 1990a). Farm history In January 1985 a severe outbreak of theileriosis occurred at Willsbridge Farm (grid ref. TR5890), especially among weaners. In previous years the farm had also lost animals during the rainy season, but theileriosis had not been confirmed. Theileria parva bovis-infected cell lines were established from three sick animals and these were tested for their binding capacity of specific monoclonal antibodies (Irvin et al., 1983 ). They were found to be typical for T. parva (Koch et al., 1988 ). In January 1986, five bait animals were exposed at Willsbridge Farm. They became infested with thousands of adult R. appendiculatus ticks. Within 3 weeks all five animals had become sick, and four died oftheileriosis. Batches of nymphs were applied on three of these animals for isolation of the parasite as described.

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Immunization and field challenge The weaners were immunized, in early November 1986, by subcutaneous inoculation of 1.0 ml of a 1 : 10 dilution of sporozoite stabilate in front of the parotid lymph node. Fifteen animals were immunized with the Boleni stock and 15 with a mixture of the three Willsbridge stabilates. No chemoprophylactic treatment was used. Rectal temperatures were recorded every other day, starting 9 days after infection. At the same time biopsies were taken from the regional parotid lymph node, stained with Giemsa's stain and examined. Two months later, at the start of the theileriosis season the animals were moved to Willsbridge Farm, together with the 10 controls. Rectal temperatures were recorded every other day, until animals showed enlargement of lymph nodes, then recording was done daily. A temperature above 39.5°C was taken as an indication of pyrexia. Blood smears were prepared from the tail tip every second day, until fever or swollen lymph nodes were detected, then daily. Biopsies were taken from the parotid lymph nodes when they became enlarged and from animals with pyrexia. As soon as schizonts were detected, all animals were sampled daily. Blood and lymph node smears were stained with Giemsa's stain and examined at the farm. Blood smears were examined once per month following recovery and throughout the next year. In the second theileriosis season (January-March 1988) the controls from the first season and the immunized animals were monitored (temperatures, blood and lymph node smears) every fortnight, while temperatures, lymph node and blood smears were taken every other day from the eight new control animals. No acaricidal treatment was applied during exposure at Willsbridge Farm. Six experimental animals were selected at random and all ticks on the left ear were counted weekly. The weights of the 10 control animals and 15 immunized animals selected at random (seven Boleni immunized and eight Willsbridge immunized) were recorded regularly during their stay at the farm. Sera were collected every fortnight during the period of the immunization and field challenge. In the following dry season sera were taken four times and in the second wet season sampling was continued at monthly intervals. The antibody titres were measured with the immunofluorescent antibody test (IFA test, Burridge and Kimber, 1972) using T. parva boris (Berea stock, P.D. Cockroft and B. Byron, personal observation, 1983) schizont antigen. Selected serum samples were retested with piroplasm antigen of T. parva and T. taurotragi. Clean nymphs of R. appendiculatus were applied in ear bags to reacting animals during exposure. After the nymphs had attached, the ear bags were taken off, but were replaced as engorged nymphs started to drop off. After

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189

moulting 50 pre-fed adults from each batch were dissected to assess infection rates. Three batches of adult ticks from nymphs applied to three immunized animals during the first Theileria infection (see Results ), were tested at the Department of Tropical Veterinary Medicine and Protozoology, Utrecht. Each batch was used to infect one of three splenectomized Friesian calves (identification nos. 469, 471 and 472 ). Reactions were recorded by daily monitoring of temperature, blood and lymph node smears and clinical reactions. Piroplasm antigen was prepared and serological titres to this antigen and to control antigens of T. parva and T. taurotragi were compared in an IFA test with positive control sera from these species and sera from the infected animals. RESULTS

Reactions to immunization The stabilate dose of 0.1 ml caused severe reactions. Three Boleni- and two Willsbridge-infected animals had to be treated with parvaquone (Clexon, Wellcome). One of the Boleni-infected animals was treated after a relapse. Another Boleni-infected and treated animal died as its treatment started too late. Records were kept of the order in which the animals were inoculated (not shown). No relationship was found between the severity of the reaction and the order of inoculation. In other words, the time interval between thawing of the stabilate and inoculation (maximum 45 min) did not affect the disease response.

Field exposure In the first season tick numbers were low. The highest numbers of ticks were found after 28 days in the field; the mean of six animals was 48 per ear. This number does not differentiate instar, sex or species; however, the majority of the ticks found on the ears in January-February are R. appendiculatus adults (R.A.I. Norval, personal observation, 1984). In the second outbreak season, the mean of eight animals was 34 per ear at its maximum in the last week of January. The reactions to infections with Theileria parasites in the first theileriosis season were divided into two distinct periods, with very little overlap. They can thus be designated as the first and the second Theileria wave (Table 1 ). During the two waves, three other blood parasites, affecting the cattle to a variable degree (never seriously), were recorded: Babesia bigemina, Borrelia theileria and Eperythrozoon sp. Although two to four different parasites were sometimes detected simultaneously, it was generally possible to relate tern-

13

5 2

7(10)

2(14) 6(15)

2

3(15)

14(15)

11 (14)

8(10)

72

8~

6.5

1One additional animal was positive for schizonts continuously from the first wave until Day 41. 2One additional animal was positive for schizonts continuously from the first wave until Day 30. The total number of the group is given in parentheses.

2.5

7(14)

Boleni immunized Willsbridge immunized

3

4(10)

Control animals

1(15)

2(14)

7(10)

Animals with schizonts

Mean days pyrexia

Mean days schizonts

Animals with pyrcxia

Animals with schizonts

Animals with pyrexia

Mean days pyrexia

Second wave: Days 25-51 after exposure

First wave: Days 14-23 after exposure

Reactions of control and immunized animals to two subsequent Theileria infections

TABLE 1

_2

2~

11

Mean days schizonts

one killed in extremis, one died

Remarks

t-

7: ©

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191

perature reactions to only one of them. Babesia and Borrelia infections were usually treated with imidocarb dipropionate (Imizol, Cooper) and with penicillin (Tricil, CAPS) respectively, to control the temperature reactions caused by these parasites that might conceal a Theileria reaction. All cattle developed swollen parotid lymph nodes 10-14 days after exposure on Willsbridge Farm. This represented the first wave of infection. The temperatures did not rise significantly above 39.5°C, except in one animal, which showed a temperature peak of 41 °C on Day 16. Control and immunized animals reacted similarly. Four of 10 controls and 10 of 29 immunized animals had temperature reactions. Eight of 10 controls and 25 of 29 immunized animals had schizonts (Table 1 ). The time period and duration of reactions was the same for i m m u n i z e d and non-immunized animals. None of the reacting animals showed signs of serious clinical illness. The second Theileria wave affected the non-immunized controls in a surprisingly synchronized way. Seven out of 10 animals developed schizonts and showed temperature reactions. One control animal was killed in extremis and another died, both oftheileriosis (Table 1 ). During the same period two Boleni-immunized animals were found to have schizonts. One had schizonts for 2 days without fever. The other, which had a continuous schizont parasitosis from the first wave, had fever for 7 days, but the temperature dropped immediately after treatment of a concurrent Borrelia infection. One Willsbridge-immunized animal had continued to show schizonts since the first infection. No clinical signs were observed, except for 6 days pyrexia. Five other Willsbridge animals had transient temperature reactions without schizonts. Some susceptible cattle, which were serologically positive to T. parva bovis schizont antigen before immunization or challenge, had titres as high as 1 : 640 - only a two-fold dilution below a titre assumed to be typical for T. parva bovis. The serologically positive animals reacted similarly to the immunization as the negative ones. Some of those serologically positive for T. taurotragi reacted again to the parasite of the first Theileria infection and the immunized animals were also susceptible to that Theileria parasite. Control animals infected during the first wave reacted to the second wave similarly to the ones in which no schizonts had been seen during the first wave. Of the three tick batches that engorged as nymphs on the immunized animals during the piroplasm parasitaemia of the first Theileria wave, two transmitted T. taurotragi and one T. parva. Infection rates of the tick batches and reactions of the animals are given in Tables 2 and 3. The splenectomized calves 469 and 471 were infested with 104 and 200 adults, respectively, and had mild clinical reactions. Schizonts were numerous, especially in 469. The first piroplasms appeared 2-4 weeks after the schizonts had disappeared, much later than some of the field-infected weaners. Sufficiently high numbers of piroplasms were eventually found to prepare piroplasm antigens and these were identified as T. taurotragi, using positive

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TABLE 2 Attempted isolation of the first wave Theileria sp. Calves infested with tick batches engorged during the piroplasm parasitaemia of the first Theileria challenge Calf no.

No. of ticks applied

Pyrexia, first day, duration in days

Mean number infected acini per tick

Schizonts, first day, number of days max. frequency L

Piroplasms, first day, max. parasitaemia 2

469 471 472

104 200 240

D13,4 D13,4 **4

10 26 70

D9,12, + + + D9,16, + DI0,21, + +

D35 -3, + + D66 -3, + DI9-D30, + +

~Maximum frequency of schizonts: +, one schizont per many fields; + +, up to one schizont per field: + + +, more than one schizont per field. 2Maximum parasitaemia of piroplasms: +, < 1% of erythrocytes infected; + +, 1-10% of erythrocytes infected. 3parasitaemia continued until the end of the experiment (4 months later). 4Calf no. 472 suffered irregular fever attacks, not clearly related to the Theileria infection. It died on Day 30. TABLE 3 Reciprocal sera-antigen titres of three calves infected with the first wave Theileria isolales Sera

Negative

7~p. parva

Piroplasm antigens

Calf no.

T.p. parva

T. taurotragi

469

( Pugu ) l

( Lawfield )2

471

472

40 10240

40 320

40 320

neg 320

neg 5120

40

2560

2560

2560

ncg

( 640 )4

1280

2560

2560

( 160 )4

(640) 4

1280

2560

2560

(160) 4

80

neg

neg

neg

( Burenga ) 3

7; taurotragi (Chiltington)2 Calf 469 (Day 108) Calf 471 (Day 122) Calf 4725 (Day 27)

neg

~Tanzania (Uilenberg et al., 1977a). 2Zimbabwe (Lawrence and MacKenzie, 1980; Uilenberg et al., 1982). 3Rwanda (R.W. Paling, unpublished data, 1980). 4Faint fluorescence in all positive dilutions. 5Calf472 died on Day 30, before significant antibodies could develop.

control sera for both species (Table 3). Antibody titres developed late. Although piroplasms appeared 25 and 66 days after infection, the highest titres to T. taurotragi piroplasm antigen were found at 3-4 months. Moderate titres to T. parva piroplasm antigen had developed at the same time, but the fluorescence was faint at all dilutions in contrast to that of 7: taurotragi antigen.

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Calf 472, infested with 240 ticks, had an intercurrent broncho-pneumonia which probably contributed to its death. It died before it developed antibodies, but a piroplasm antigen was prepared. Theileria parva (Burenge) sera reacted strongly with this antigen, while T. taurotragi (Chiltington) sera did not and sera from 469 and 471 only gave a faint reaction at low dilutions. It is possible that the weaner on which the tick batch engorged, which infected calf 472, was still a carrier of the immunizing parasite. In the Giemsa stained blood smears as well as the piroplasm antigen in the IFA staining technique, T. taurotragi piroplasms appeared to be larger, showing more broad, oval shapes than the T. parva piroplasms. At the time the cattle moved to the farm, the mean weight was 17 kg higher for the control animals (range 135-190 kg) than for 15 randomly selected immunized animals (range 95-200 kg). The difference in weight between the groups was a result of the severe reactions to the immunization. One of the immunized animals was clearly stunted, while a few others never seemed to develop well. During the second wave, when severe reactions occurred in the control group, the difference in weight between the controls and the immunized animals diminished to an insignificant level (when the stunted animal was excluded). In the second year of exposure four out of eight new control animals had rather mild reactions, and four had severe reactions, fatal in one. During the same period one of the previously exposed control animals had a clinical reaction. It had not reacted to the second Theileria wave in the first year. DISCUSSION

Both the T. parva bovis Boleni and Willsbridge stabilates protected well against natural challenge at Willsbridge Farm. In an earlier trial these two isolates had been found to protect against each other. Three other isolates from different parts of Zimbabwe were also found to be cross-protective with the Boleni isolate (Koch et al., 1988, 1990a). It is expected that immunological differences between strains may not be as important in the epidemiology of January disease in Zimbabwe as in East Coast fever areas. The risk of infection with Theileria parva lawrencei is also limited as buffalo are restricted to certain areas and farms. Theileria taurotragi and T. parva boris apparently behave similarly in the bovine host. As they also have a common vector, mixed infections could be assumed. Those mixed infections could not be demonstrated in this trial; immunized animals were still susceptible to T. taurotragi. However, even animals serologically positive to T. taurotragi developed a parasitosis during the first wave. Apparently these cattle became infected again with T. taurotragi. It could be explained by assuming the existence of immunologically different

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T. taurotragi types, although so far there is no experimental evidence for this hypothesis. Some experimental cattle used in this trial had titres to the T. parva bovis (Berea) schizont antigen and yet they were susceptible when challenged with T. parva bovis (Boleni and Willsbridge). Cross-reactions between T. parva and T. taurotragi in the IFA test, using T. parva piroplasm and schizont antigens, have been described (Burridge et al., 1974; Uilenberg et al., 1977b; de Vos and Roos, 1981; Uilenberg et al., 1982; Jongejan et al., 1986 ). While low titres had been found in those studies, higher titres to T. parva schizont antigen owing to T. taurotragi infection have been found in the present study. As the routine IFA test is not entirely specific for T. parva, there is no way at present of reliably assessing the susceptibility of an individual animal other than by a controlled challenge. When studying the epidemiology of January disease at this stage, it is not possible to assess infection in a particular area other than by recording outbreaks. The reactions to immunization were too severe to be acceptable. As the susceptibility of herds and individuals varies considerably, immunization with chemoprophylactic treatment (Radley et al., 1975; Brown et al., 1977; Radley, 1981 ) may be necessary to make vaccination safe under all circumstances. More experiments are necessary. The immunization produces carriers (Koch et al., 1990b). The incidence of carriers after immunization might be many times larger than after natural outbreaks, therefore m o v e m e n t restrictions of immunized cattle will have to be considered. The main benefits from immunization would be a reduction in losses owing to clinical theileriosis on chronically infected farms and a reduction in dipping frequency. It would allow a new approach to tick control based on economic considerations such as cost of dipping and production loss caused directly by the tick. When tick control is relaxed, diseases other than theileriosis can occur (Uilenberg, 1985 ). The area in Zimbabwe affected by T. parva bovis is to a large extent free from Amblyomma hebraeum and Amblyomma variegatum (Norval, 1983; Norval et al., 1985 ) and therefore free from heartwater and T. mutans infection, which have complicated immunization trials in other countries. Precautions are necessary to ensure that it will remain free of Amblyomma ticks. ACKNOWLEDGEMENTS

The work has been sponsored by the Danish Aid Organisation (DANIDA) through the Food and Agricultural Organisation (FAO). We are most grateful to Mr. F.M. Fussell, owner of Willsbridge Farm, for his help and understanding during the three years we undertook trials on his farm. We also thank Professor Dr. J.A. Lawrence for his comments on this paper.

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Radley, D.E., 1981. The infection and treatment method of immunization against Theileriosis. In: A.D. Irvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff, The Hague, pp. 227-237. Radley, D.E., Brown, C.G.D., Cunningham, M.P., Kimber, C.D., Musisi, F.L., Payne, R.C.,

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Purnell, R.E., Stagg, S.M.G. and Young, A.S., 1975. East Coast fever: 3. Chemoprophylactic immunization of cattle using oxytetracycline and a combination of theilerial strains. Vet. Parasitol., 1: 51-60. Short, N.J. and Norval, R.A.I., 1981. Regulation of seasonal occurrence in the tick Rhipicephalus appendiculatus Neumann 1901. Trop. Anim. Health Prod., 13:19-24. Thomson, J.W., 1985. Theileriosis in Zimbabwe. In: A.D. Irvin (Editor), Immunization against Theileriosis in Africa. International Laboratory for Research on Animal Diseases, Nairobi, pp. 48-57. Uilenberg, G., 1976. Tick-borne livestock diseases and their vectors. 2. Epizootiology of tickborne diseases. World Anim. Rev., 17: 8-15. Uilenberg, G., 1985. Possible impact of other tick-borne diseases following East Coast fever immunization. In: A.D. Irvin (Editor), Immunization against Theileriosis in Africa. International Laboratory for Research on Animal Diseases, Nairobi, pp. 118-122. Uilenberg, G., Schreuder, B.E.C., Mpangala, C. and Tondeur, W., 1977a. Studies on Theileriidae (Sporozoa) in Tanzania. IX. Unidentified bovine Theileriae. Tropenmed. Parasitol., 28: 494-498. Uilenberg, G., Silayo, R.S., Mpangala, C., Tondeur, W., Tatchell, R.J., Sanga, H.J.N., 1977b. Studies on Theileriidae (Sporozoa) in Tanzania. X. A large-scale field trial on immunization against cattle theileriosis. Tropenmed. Parasitol., 28: 499-506. Uilenberg, G., Pieri6, N.M., Lawrence, J.A., de Vos, A.J., Paling, R.W. and Spanjer, A.A.M., 1982. Causal agents of bovine theileriosis in Southern Africa. Trop. Anim. Health Prod., 14: 127-140.