Immunization against east coast fever: The use of selected stocks of Theileria parva for immunization of cattle exposed to field challenge

Veterinary Parasitology, 23 (1987) 2341 Elsevier Science Publishers B.V., Amsterdam

23 - Printed

in The Netherlands

IMMUNIZATION AGAINST EAST COAST FEVER: THE USE OF SELECTED STOCKS OF THEZLERZAPAR VA FOR IMMUNIZATION OF CATTLE EXPOSED TO FIELD CHALLENGE

S.P. MORZARIA, A.D. IRVIN, E. TARACHA’ T. FUJINAGA and J. KATENDE International Laboratory Nairobi (Kenya) (Accepted

for publication

, P.R. SPOONER,

W.P. VOIGT,

for Research on Animal Diseases (ILRAD), 4 February

P.O. Box 30709,

1986)

ABSTRACT Morzaria, S.P., Irvin, A.D., Taracha, E., Spooner, P.R., Voigt, W.P., Fujinaga, T. and Katende, J., 1987. Immunization against East Coast fever: the use of selected stocks of Theileria parva for immunization of cattle exposed to field challenge. Vet. Parasitol.. 23: 23-41. Two antigenically different stocks of Theileria parva parva (Kilifi and Marikebuni), previously characterized as belonging to groups A and C respectively on monoclonal antibody (MAb) profiles, were selected for immunization of different breeds of cattle against East Coast fever (ECF) by the infection and treatment method. A total of 52 immunized cattle and 33 susceptible controls of different group sizes were exposed to field challenge by ticks for periods of 42-90 days at three field sites where ECF is endemic on the Kenyan coast. All immunized cattle survived ECF challenge, but 87% of the controls died of the disease. The cattle exposed at one site had been immunized 1 year earlier and maintained tick-free in the intervening period. The level of immunity in these cattle was similar to that of cattle which had been immunized 1 or 2 months prior to exposure. Thus, immunity had not waned over the l-year period. A study at another site showed that acaricidal treatment of immunized cattle could be safely extended from twice a week to once every three weeks, whereas in susceptible cattle even twice weekly spraying did not control ECF. The isolates made from infected controls during the trials indicated the presence of three T. p. parva stocks as defined by MAb profiles. Of the two stocks used for immunization, T. p. parva Marikebuni induced broader protection. In view of the apparent limited antigenic diversity of T. p. parva strains within the Coast Province it is suggested that the Marikebuni stock might represent a key stock for vaccination in this area.

INTRODUCTION

Theileriosis of cattle, principally caused by the protozoan parasites Theileria parva pm-m (East Coast fever) and T. p. lawrencei (Corridor disI Seconded

from Veterinary

0304-4017/871803.50

Research

Laboratory,

Kabete,

0 1987 Elsevier Science

Kenya.

Publishers

B.V.

24

ease), is a major constraint to livestock development in East and Central Africa. The vector involved in transmission is the three-host tick Rhipicephalus

appendiculatus.

Of the several methods of immunization which have been investigated (Irvin and Morrison, 1985), immunization by infection and treatment, or chemoprophylaxis (Radley et al., 1975a), shows promise for use in the field. This method involves inoculation of the host with live 7’. parua sporozoites in the form of cryopreserved tick-derived stabilate (Cunningham et al.: 1973) and simultaneous treatment with a long-acting formulation of oxytetracycline (Radley, 1978). Cattle thus immunized are found to be immune to homologous challenge but may not be fully protected against heterologous challenge. The presence of different strains of 2’. parua (Young et al., 1973; Radley et al., 1975a, b; Dolan et al., 1980) is therefore a complicating factor in successful immunization of cattle in the field. Until recently the only method of differentiating strains of T. parua was to carry out cross-immunity trials in cattle; such trials are expensive, laborious and not always reliable. More recently a method has been developed which detects antigenic diversity of T. parua using monoclonal antibodies (MAbs) raised against the macroschizont stage of the parasite (Pinder and Hewett, 1980). MAb profiles, which can thus be determined for different theilerial stocks, provide an in vitro method of comparing the antigenic composition of these stocks (Minami et al., 1983) and theilerial stocks showing similar profiles can be grouped together. Furthermore, Irvin et al. (1983) showed that stocks of T. p. parua within the same MAb group were cross-protective in cattle, whereas stocks from different groups might not crocs-protect. The correlation between in vivo cross-immunity and in vitro MAb tests indicated that MAb profiles might provide an additional tool for detecting strain differences in T. p. parua. This work also suggested that MAb profiles in conjunction with in vivo cross-immunity tests might be used for selecting appropriate key stocks for immunization of cattle against ECF under field conditions. In order to test the above suggestion we selected two stocks of T. p.parua, Kilifi and Marikebuni, as key stocks for immunization of cattle in the field and then exposed the immunized cattle to natural tick/parasite challenge in areas near the sites from where these or related stocks were originally isolated. The selection of the above stocks was based on the MAb test and cross-immunity trials (Irvin et al., 1983) which showed that all of the several stocks originating from the Coast Province of Kenya belonged to one of three groups (A, B and C, Minami et al., 1983), and that the Kilifi and Marikebuni stocks (Groups A and C respectively), when used together, provided the widest protection. The trial sites selected were all in the Kilifi District of the Coast Province of Kenya from where the immunizing stocks originated. The main reasons for selecting these sites were that ECF is endemic in the area and buffalo are not present; thus, Corridor disease due to T. p. lawrencei which can cause complications in immunizing cattle against ECF, is absent.

25 MATERIALS

AND METHODS

Trial sites Three farms were selected on the basis that ECF was a known problem and a significant constraint to cattle rearing in the respective areas. All farms were in the Kilifi District of the Coast Province of Kenya; their location is given by the standard grid reference taken from the 1: 250 000 map of the District (Survey of Kenya, 1972). Since all the farms selected seek to control ticks on their own cattle by regular spraying or dipping with acaricides, trial cattle were held in fields on the edge of each property and driven daily into adjacent communal grazing areas, where tick control was minimal. The sites were as follows: (i) Mtwapa (EF6583), 20 km north of Mombasa and 10 km from Junju, from where a T. p. parua group A stock was isolated (Minami et al., 1983). (ii) Kiswani (FG5016), 10 km north west of Malindi and 8 km south of Marikebuni, from where a T. p. parua group C stock was isolated (Minami et al., 1983). (iii) Kibarani (FG0294), 50 km north of Mombasa and 10 km north of Mavueni, from where a T. p. parua group B stock was isolated (Minami et al., 1983). Cattle Different

breeds of cattle were used at the three farms as follows:

Mtwapa Eleven Jersey bull calves, 3-12 months of age, were purchased from the farm on which the trial was conducted. Eight were immunized and three were non-immunized controls. Kiswani Twenty-five 4.5-month-old Sahiwal/Red Poll calves were purchased from the farm on which the trial was conducted. Eighteen were immunized and seven were controls. Survivors from this experiment, together with 12 additional controls of similar age, were subsequently exposed to further field challenge in a second phase of the experiment. Kibarani Two trials were carried out at immunized 12 months earlier and ILRAD’s experimental farm and from Kiswani (above) were used. Borans, the susceptible Borans months, 18 months and 6 months

this site. In the first, 22 Boran cattle (17 five susceptible controls) originating from five Sahiwal/Red Poll crosses purchased At the time of exposure the immunized and the susceptible crosses were 13-18 old, respectively.

26

In the second trial, 25 Borans (12 immunized and 13 susceptible controls) purchased from a commercial farm 20 km south of the trial site were used. At the time of field exposure they were 814 months of age. Tick infestations were controlled by regular acaricidal spraying prior to the field exposure. Cattle were monitored for serum antibodies to T. parua schizont antigen using the indirect fluorescent antibody (IFA) test as described by Goddeeris et al. (1982). All animals were negative for antibodies prior to immunization and the controls remained negative until the time of challenge. Parasite stocks for immunization

Cryopreserved stabilates (Cunningham et al., 1973) of T. p. parua (Kilifi) and T. p. parua (Marikebuni) (Irvin et al., 1983) were used either separately or combined as described below. Stabilates were injected, subcutaneously below the ear, immediately after thawing and dilution with medium used for cryopreservation (Cunningham et al., 1973). Immunization

protocol

The majority of the cattle were immunized by the infection and treatment method described by Radley et al. (1975a). Cattle were inoculated with a long-acting formulation of oxytetracycline (Terramycin/LA, Pfizer Ltd, Sandwich, Kent, U.K., injectable solution, 200 mg ml-‘) intramuscularly (im) at a dose of 20 mg kg-’ body weight (bw) and immediately afterwards inoculated subcutaneously (SC) with a lethal dose of the chosen stabilate of T. p. parua. The exceptions were the two cattle in the Kiswani trial and the three cattle at the Kibarani trial (see below). The immunization procedure at the different sites was as follows. Mtwapa

On Day 0, all eight calves were immunized with T. p. parua Kilifi and Marikebuni. The three controls received oxytetracycline treatment alone. Twenty-four days later all the immunized calves were given a secondary immunization. Four of these calves received stabilates only and the other four received the same concentration of the stabilates plus concurrent oxytetracycline treatment as before. Kiswani

On Day 0, 16 calves were immunized with T. p. parua Marikebuni stock. Seven controls received oxytetracycline only. In addition, two calves were each inoculated with the stabilate alone to test its infectivity. These two calves reacted severely; they were treated with Clexon (Wellcome Foundation Ltd., London, U.K.; one treatment of 10 mg kg-’ bw intramuscularly) and recovered. They were subsequently included with the group of 16 immunized cattle, giving a total of 18 in this group.

27

During primary immunization 14 of the 16 cattle showed macroschizonts and/or temperature > 39.5”C, starting between 6 and 19 days after immunization. At this time there was a danger of the farm becoming cut off by floodwater, making it potentially impossible to monitor the cattle; in order to obviate the risk of any severe reactions developing, 14 of the 16 cattle were treated with Clexon as above on Day 19. All immunized cattle were clinically normal and macroschizonts were not detectable by Day 25 post-immunization. Kibarani In the first trial, 17 of the cattle sent to Kibarani had been immunized at ILRAD 12 months earlier. Fourteen were immunized by infection and treatment using T. p. parua (Marikebuni) stabilate and a simultaneous treatment with oxytetracycline as above. Each of the 14 cattle was then given a potentially lethal homologous challenge 6 weeks later to which they were fully resistant. In addition, three similar cattle which had recovered naturally from T. p. parua (Marikebuni) stabilate-induced infection, were included with the 14 immunized animals, giving a total of 17 in this group. The 10 controls received oxytetracycline alone. Just prior to sending these cattle to Kibarani, large numbers of ECF-free nymphal R. appendiculatus were applied to four of them and, although piroplasms were not detectable in the cattle, three of the four groups of resultant adult ticks transmitted fatal ECF to susceptible cattle thus demonstrating that at least three of the previously immunized cattle were still carrying infective piroplasms at the time of field exposure. In the second trial, carried out 12 months later, each of the 12 Boran cattle was immunized using T. p. parua Marikebuni stabilate and a concurrent treatment with oxytetracycline. By Day 18 nine cattle had shown a mild transient ECF reaction and recovered. The three exceptions showed persistent hyperthermia and parasitosis for 5 days and were treated with Clexon as above. All cattle were normal by Day 20 post-immunization. The 13 controls received oxytetracycline alone. Field exposure When exposure began, tick control was stopped for the period of the trial, and cattle were taken daily from their respective holding paddocks to graze in adjacent communal grazing amongst local cattle which were not dipped or sprayed. The length of the exposure period at Mtwapa was 63 days, commencing 80 days after initiation of immunization; at Kiswani cattle were exposed for 90 days, starting 49 days after immunization. At Kibarani in the first trial, exposure lasted 42 days, starting approximately 12 months after immunization, and in the second trial exposure lasted for 83 days, starting 20 days post-immunization (Table I).

28 TABLE I Summary of the immunization immunization trials

procedures and exposure period of cattle in four ECF

Mtwapa

Kiswani

Kibarani I

Kibarani II

Cattle breed

Jersey

Sahiwal/Red Poll cross

Boran

Boran

No. immunized cattle

8

18

17

12

No. controls

3

7

10

13

Immunizing stock

Kilifi/ Marikebuni

Marikebuni

Marikebuni

Marikebuni

Additional treatments

Booster stabilate

Clexon

(I4Y

Booster stabilate

Clexon (3)

Days to exposure after immunization

80

49

370

20

Duration of exposure (days)

63

90

42

83

Time of exposure

Dec.-Feb.

Jan.-March

April-May

May-July

MAb profile of challenging parasites

C

B

A

NDb

a Figures in brackets indicate the number of cattle treated. bNot done.

When the first phase of the Kiswani trial was completed, surviving cattle were returned to the usual twice-weekly spraying regimen for 5 months; they were then divided into two groups, each of eight animals. These cattle, together with two groups each of six susceptible control cattle, were then exposed to further field challenge as before and a modified spraying regimen was introduced: one immunized group and one control group were sprayed once every 3 weeks, and the other two groups were sprayed twice a week. The acaricide used was Delnav (Wellcome, Kenya Ltd.). This second exposure period lasted 167 days, from late September to early March. All of the immunized cattle exposed at Mtwapa, Kiswani and in the second tial at Kibarani were positive for 2’. parua antibodies when field exposure began; of those exposed in the first trial at Kibarani, six of the 17 were positive, i.e. antibody titres had waned in 11 of these cattle following immunization 12 months earlier. Sampling

of cattle

During immunization and field exposure all cattle were examined daily (every other day during Phase 2 of the Kiswani trial), rectal temperatures

29

were recorded and lymph nodes palpated. When the temperature of an animal rose above 39.4% (regarded as a febrile response), lymph node biopsy smears were prepared from parotid and prescapular lymph nodes. Smears were fixed in methanol, stained with Giemsa’s stain and examined for the presence of theilerial schizonts. Blood smears were prepared from ECF-positive cattle and similarly stained and examined for piroplasms. Where other blood or tick-borne infections were suspected (especially babesiosis, anaplasmosis, or trypanosomiasis) appropriate samples were taken and examined. Blood was collected weekly from all cattle for separation of serum and for measurement of erythrocyte packed cell volume percent (PCV). Sera were screened by the IFA test for the presence of antibodies to T. paruu, T. mutans, and Trypanosoma spp., and with the card agglutination test kit (Hynson, Westcott and Dunning, Baltimore, MD) for antibodies to Anaplasma marginale. During field exposure, the numbers of rhipicephalid ticks (mostly R. appendiculatus) on the ears and head of all cattle were recorded every other day. The presence of other tick species elsewhere on the body was also noted but counts were not made. Weights of cattle were recorded once a week throughout the entire period of the Kiswani trials. Appropriate weighing facilities were not available at the other sites. All cattle that died were subjected to post-mortem examination and appropriate samples were collected to determine the cause of death. Parasite isolations were made from at least one of the controls during its ECF reaction at each of the trial sites: lymph node biopsies were taken for establishment of parasites in culture (Brown, 1979), and clean nymphal R. appendiculatus were applied in ear bags to pick up parasites from the blood (Bailey, 1960). Monoclonal antibody profiles of parasites isolated in culture were determined (Minami et al., 1983). Stabilates were prepared from the adult ticks derived from nymphs fed on control calves (Cunningham et al., 1973). These were kept as reference stocks or used to infect cattle for derivation of further cultures for MAb profile testing. A summary of the immunization procedures and exposure periods in all 4 trials is presented in Table I. 0 ther treatments All cattle were vaccinated against foot and mouth disease within 3 months prior to the start of trials. A number of cattle contracted anaplasmosis or trypanosomiasis; these were treated respectively with one dose of Imizol (12% w/v Imidocarb dipropionate, Wellcome, Kenya Ltd., Nairobi) at the rate of 1 ml 100 kg-’ bw given SC, or with one injection of Berenil (Hoechst AG, Frankfurt, FRG) given im at a dose rate of 7 mg kg-’ bw. Cattle exposed in the second

30

Kibarani trial were treated with Samorin (May and Baker, Dagenham, U.K.) at the rate of 1 mg kg-’ bw given im as a prophylactic against trypanosomiasis. During this phase of of the trial a number of the cattle contracted lumpy skin disease. In all cases this cleared spontaneously. Statistical

analysis

Data on tick counts, PCVs and animal weights were analysed using 2 factor experimental design with repeated measures (Winer, 1971). The test of significance was carried out at 5% or less. RESULTS

Mtwapa On primary immunization, all the calves showed inapparent or mild reactions (transient parasites and/or febrile response). However, following booster immunization, one calf which received stabilate alone reacted more severely and was treated with Clexon. The results of field exposure are shown in Fig. 1. All three controls died

i i........ i..............

0

I

I

I

I

I

10

20

30

40

50

60

Days after exposure Fig. 1. Survival Mtwapa.

of

immunized

and

control

cattle

(Jerseys)

to

ECF

field

challenge

at

31

of ECF between Days 47 and 58. None of the immunized cattle showed clinical ECF reactions but three showed transient theilerial parasitosis between Days 37 and 49. During the exposure period, tick challenge increased rapidly and reached a maximum of 115 (mean number of counts from all cattle). No significant difference in tick challenge was observed between the two groups. One control and three immunized calves were successfully treated for T. uiuux infection. Parasites were isolated in culture from two controls; these were both classified as type C on MAb profile testing, the same as the T. parua (Marikebuni) used for immunization. The newly-isolated stocks were named T. parua (Mtwapa 1 and 2). Kiswani The reactions of the cattle, on primary immunization, have been described above (see Materials and Methods). The results of field exposure are shown in Fig. 2. Five controls reacted severely and four died of ECF; the fifth animal was treated with Halofuginone (Hoechst AG, Frankfurt, FRG). The drug was used because it does not affect piroplasm parasitaemia and thus

......

control

C.W.

20-

16 -

aa E 5 ”

12-

z t 3

- 40

6

6,................................:

i i.... :.......................................,......................:

4

-

I.. .... . . ...i :............

. 0

6

40

50

60

70

60

-

20

60

Days after exposure Fig. 2. Survival of immunized and control cattle (Red Poll/Sahiwal croaaea) to ECF field challenge at Kiawani. The shaded area represents deaths due to Heartwater.

32

enabled us to make parasite isolation by tick pick-up. The animal recovered after prolonged convalescence. The other two controls showed mild ECF reactions and recovered. Two of the controls which died showed mild transient parasitosis about 3 weeks before the subsequent fatal reactions. Eight of the immunized cattle showed mild transient ECF reactions (one of them on two occasions). The other 10 immunized animals showed no such reactions. However, three immunized calves died of heartwater (Cowdria ruminantium infection); another animal appeared to recover spontaneously from the disease. During the exposure period, tick challenge increased progressively and reached a maximum (mean 105.4) just before the termination of the exposure. Intensity of tick infestation was similar in both groups. Most of the cattle showed T. mutans and A. marginale infections which caused moderate anaemia by the end of the exposure period. The mean PCVs of the immunized and control groups are shown in Table II. Because of anaemia all survivors were treated with Imizol on termination of the trial. No trypanosome infections were detected. TABLE

II

Erythrocyte in the Coast Trial* site

PCV (W) in ECF-immunized Province of Kenya Number animals

of

and control

Previous history

cattle

exposed

to natural

challenge

PCV (%) Start of exposure

End of exposure

f 3.0 f 3.0

23.3 22.5

+ 2.3 r 2.1

7 (S/R)

Immunized Controls

_.31.8 29.8

KIBARANI I

17 (B)b 5(B) 5 (S/R)

Immunized Controls Controls

31.4 34.2 34.6

2 3.8 f 2.9 f 2.9

23.9 21.0 21.0

f 3.5 * 3.7 + 3.5

KIBARANI II

12(B) 13(B)

Immunized Controls

29.3 31.9

2 3.7 + 2.7

20.0 20.7

+ 3.3 + 5.2

KISWANI

18 (S/R)a

a(S/R) = Sahiwal/Red Poll cross. b(B) = Boran. *PCVs were not determined at Mtwapa.

Clean ticks were applied to one of the controls which became infected. These were subsequently used to infect a susceptible calf. A culture was established from this calf and the parasite shown to be group B on MAb profile. By the time the second phase of the Kiswani trial began, the immunized cattle were fully recovered from the effect of their previous exposure. In

33

this phase of the trial, all the control cattle being sprayed once every 3 weeks contracted severe ECF: five died and one recovered. Of the control cattle being sprayed twice a week, four died of ECF; the other two showed no clinical or parasitic reaction but developed antibodies to T. parua, as demonstrated in the IFA test. None of the immunized cattle in either group showed any signs of ECF and all survived the 5.5month exposure period. The results are summarised in Table III. TABLE III Results of field challenge of ECF-immunized ent spraying interval schedules Spraying interval

Cattle No.

Once every

3 weeks Twice weekly

and control cattle maintained under differ-

ECF reactions

Mortality (%I

Previous history

NRa

SRb

Death

ECF-immunized Susceptible controls

8 0

0 6

0 5

0 83

ECF-immunized Susceptible controls

8 0

0 4

0 4

0 66

*No reaction. bSevere reaction.

Kibarani

The results of the first trial are shown in Fig. 3. All 10 controls died of ECF, with mean times to death of 34.8 days for the Sahiwal/Red Polls and 34.3 days for the Borans. One Boran died on Day 51,9 days after field exposure ended. This animal underwent a prolonged and chronic ECF reaction following treatment with Clexon. Of the 14 animals artificially immunized by infection and treatment, 10 showed mild transient ECF reactions during the challenge period, as evidenced by low numbers of macroschizonts in the superficial lymph nodes. The mean duration of macroschizonts in these animals was 5.3 days in contrast to 16.1 days for the controls. One of the immunized animals died of unknown causes unrelated to ECF. Of the three immunized animals, naturally recovered following stabilate infection, all showed ECF reactions during the field exposure; two underwent mild reactions and recovered (mean duration of macroschizont parasitosis was 8 days) while one underwent a severe reaction and died. Clean ticks fed as nymphs on one infected control and one immunized animal were subsequently fed as adults on susceptible cattle to induce ECF reactions. Cultures were established from the infected cattle and characterized by MAb profiles as group A.

34

i

0

10

20

40

30

Days after

60

60

exposure

Fig. 3. Survival of immunized cattle (Borans) and control cattle (Borans Sahiwal crosses) to ECF field challenge at Kibarani (Trial I). The shaded death due to unknown causes but not ECF.

and Red Poll/ area represents

During the trial, A. marginale, T. congolense, T. vivax, T. mutans, Eperythrozoon teganodes and Borrelia theileri were detected in various cattle. As a consequence all cattle became anaemic (Table II). All survivors responded to Imizol and Samorin treatments given at the end of the exposure period. In the second trial at Kibarani none of the immunized cattle showed ECF reactions during field exposure but one died of suspected snake bite. All of the controls reacted; 11 of them reacted severely and nine died of acute ECF (Fig. 4). Two of the severely reacting animals were treated with Clexon; one recovered and the other died of chronic ECF complicated by sarcocystosis. The two remaining controls showed a mild reaction to ECF and recovered spontaneously. The tick challenge (measured up to Day 24 only) was again similar between the immunized and control groups. Intercurrent diseases observed during the challenge were trypanosomiasis, benign theileriosis (T. mutans infection) and anaplasmosis. These diseases contributed to anaemia as evidenced by low PCVs in both immunized and susceptible groups of cattle towards the end of the trial (Table II). PCVs

36

--

kmM#d

.... ..

cabol.aleD

calm

14 ..I

12

:..............{

10

I t! 0

i ........ . i.;

8

B 8

i :.......

6

..‘.............. : i

z

:

4

i...

2

0

7

30

so

40

70

60

60

Days after exposure Fig. 4. Survival of immunized and control cattle (Borans) to ECF field challenge at Kibarani (Trial II). Tick counts are not shown; these were made up to Day 24 after exposure. The shaded area represents death not due to ECF (snake bite suspected). TABLE

IV

Summarized Kenya TriaI site

results of exposure

of ECF-immunized

Reactions

Cattle No.

Previous history

a 3 18

Kiswani

7b KibaraniI

14 3 lob

Kibarani

II

12 13c

Immunized by I& Ta Controls Immunized buI&T Controls Immunized byI&T Naturally recovered controls Immunized byI&T Controls

sites in the Coast Province,

due to ECF

Mean days to Pweria

Mtwapa

cattle at three different

Mocroschizonts

Mean duration Death

Pyreria 6(3)

l(3) lO(3)

of

Macroscbiaonts

38 (3jd

42 (3)

-

46(3)

43(3)

62(3)

7(3) 4 (8)

‘I(8)

44 (8)

48 (8)

-

62(6)

68(6)

64(4)

6f6)

6f6)

21(12)

26(12)

-

16 (12)

6(12)

22(3)

26 (9)

37(l)

17(3)

ll(3)

17(D)

19(D)

34(D)

18(D)

16(D)

32 (2)

40(2)

-

2(2)

l(2)

36(11)

3701)

61(D)

aInfection and treatment method. bOne treated control not included in the statistical analysis. ‘Two treated controls not included in the statisticaI analysis. dFigures in parentheses represent number of reacting animals.

1601)

16(11)

36

returned to normal following Imizol and Samorin treatments given at the end of the trial. A summary of the survival patterns of immunized and control cattle at the three trial sites is given in Table IV. DISCUSSION

The T. p. pm-m stocks chosen for immunizing cattle at the three trial sites were selected on the basis of their MAb profiles (Minami et al., 1983) and the fact that under laboratory conditions, they gave wide cover against other stocks isolated from the Kenya Coast (Irvin et al., 1983). The selection of the Kilifi and Marikebuni stocks on the above criteria for immunization in the current trials was fully justified in that a high degree of protection was afforded to immunized cattle at all of the sites. In addition, all the theilerial isolates made from reacting control cattle at these sites fell into one of the three MAb groups previously defined by Minami et al. (1983). This finding further supports the suggestion (Irvin et al., 1983) that the number of T. p. parua strains occurring in the field may be small, and that immunization with selected stocks may give good protection over a wide area. Thus, the MAb test appears to be an effective way of characterizing T. p. pm-vu stocks from the field and selecting appropriate stocks for subsequent immunization in that area. It was interesting to note that in three trials (Kiswani, Kibarani I and Kibarani II) all cattle survived the field challenge despite having been immunized only with T. p. parua Marikebuni stock; this suggests that the Marikebuni stock might approach the type of master strain sought for immunization in ECF endemic areas (Radley, 1981). Where T. p. lawrencei from buffalo occurs (absent from this study area), good protection is harder to achieve because of the wider antigenic diversity displayed by this parasite, both on MAb profiles and in vivo (Irvin et al., 1983). In recent years a number of trials has been conducted in which cattle immunized against ECF by the infection and treatment method, have been shown to resist subsequent field challenge (Radley et al., 1975a, 1978; Robson et al., 1977; Uilenberg et al., 1977; Paling and Geysen, 1981). Similar success was achieved in the current work in that all 52 immunized cattle of different breeds survived the field challenge. Of the three animals immunized following natural recovery after stabilate infection, one died of ECF during field challenge. This was a steer in the first Kibarani trial which, as a calf < 4 weeks old, had recovered spontaneously from stabilate-induced infection 12 months earlier. It seems probable that its immunity had waned. In contrast, of the 33 controls included in the trials, 27 died of ECF, four recovered naturally and two recovered after treatment. In summary, 100% of the cattle immunized by infection and treatment survived ECF field challenge (four animals however subsequently died: three of heartwater and one of unknown causes), whereas only 12.9% of the untreated controls survived.

37

In previous studies, cattle which have recovered naturally from ECF have been shown to be resistant to challenge after 2 years (Bamett, 1957), 3.5 years (Burridge et al., 1972) and 5 years (Neitz, 1957). In the current work, cattle which had been immunized and then maintained tick-free were solidly resistant to field challenge at Kibarani 12 months later, thus showing that the quality, duration and strength of immunity generated by infection and treatment immunization, was comparable to that which follows natural recovery. In the present trials the type of challenge to which these cattle were exposed was severe and prolonged (up to 3 months without acaricidal application), and under normal management systems would be unlikely to occur. However, despite this challenge level, immunity to ECF remained solid. Inevitably other tick-borne diseases and conditions were encountered at all sites; the most serious was heartwater which caused the death of three cattle at Kiswani. Anaplasmosis and T. mutuns infections were also encountered and caused marked anaemia. Treatment with Imizol resulted in cure of the former condition, but T. mutans infections were not sufficiently severe to require treatment. The presence of T. vivax infection also necessitated treatment of some animais. In the trial at Kiswani, a number of control cattle also showed mild theilerial reactions followed by recovery and subsequent fatal ECF. Because the mild reactions failed to generate protection it is possible that a morphologically similar parasite, such as T. tuurotragi, was involved, although no isolates were made. Similar mild reactions also occurred in some of the immunized cattle at the same site. Immunization against ECF therefore, will not obviate the need to control ticks. Whatever ECF vaccination method is used, absence of tick control will result in the debilitating effects, or even death, caused by tick worry and tick-borne diseases. Therefore, some form of tick control is essential; otherwise, the effects of ticks and tick-borne diseases can negate the value of immunization. In the latter part of the trial at Kiswani, a group of immunized cattle was put onto an extended interval spraying programme and animals were sprayed once every 3 weeks as opposed to the usual practice of twice a week. There was no difference in the performance of these cattle over a 6-month exposure period in a highly endemic ECF area when compared with that of immunized cattle sprayed twice weekly. In contrast, when two groups of susceptible cattle under the same regimens were exposed in the same area, most of them died of ECF, including four of six animals sprayed twice weekly. This experiment showed that if cattle were immunized against ECF their tick control schedule could be reduced g-fold without any apparent loss of productivity and with a considerable financial saving to the farmer. It was also shown that even twice a week spraying could not adequately contain ECF in a high challenge area. Young et al. (1979) reported that maturation of T. parvu sporozoites within the tick vector could be stimulated by raising the environmental

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temperature, and infective sporozoites could be produced prior to tick feeding. The current work provides circumstantial evidence that such maturation can also occur in the field under high environmental temperatures and recently Dr. AS. Young in a joint project with us (unpublished data) has shown that a stabilate prepared from unfed ticks exposed in the field to such conditions, infected cattle with ECF. It therefore seems likely that even twice a week spraying will not adequately control ECF if ticks which are harbouring infective sporozoites succeed in attaching to the host, since transmission of infection can be expected within hours of attachment rather than a period of days which is more normal under cooler conditions where parasite maturation has to be stimulated by tick feeding. A spraying interval of once every 3 weeks worked well in the circumstances prevailing at Kiswani, since it allowed some tick feeding to occur and hence a stimulation or boosting of acquired immunity to ticks and tickborne diseases, but the schedule did not allow high or overwhelming infestations to develop which could have been deleterious to the cattle. As cattle acquire resistance to tick-borne diseases through controlled exposure as above, it may be possible to extend spraying or dipping intervals further in situations where enzootic stability is established. We are currently examining these aspects further. In particular it would be advantageous if young cattle could be immunized against ECF and then exposed to field challenge while they still retained a degree of age-related innate immunity to diseases such as anaplasmosis, babesiosis and heartwater. In this way their immunity could be boosted by challenge with less risk of pathogenic effects arising from these diseases than if the cattle were exposed when older. The question of the carrier state was recently reviewed by Dolan (1981) and by Irvin and Morrison (1985), and it appears that the condition can commonly occur (Young et al., 1981), and may be essential for the maintenance of infection in situations of enzootic stability. Fears that the carrier state may jeopardise the use of infection and treatment immunization appear groundless. The immunized cattle sent to Kibarani were in excellent condition and showed no evidence of debility or retarded growth following their immunization 12 months earlier. The piroplasm infection was extremely low and was only picked up under highly artificial laboratory conditions, which are most unlikely to occur in the field. The risk of such infection being transferred to field ticks and thence to susceptible cattle, is thus very low. Finally, it should be remembered that once immunized cattle are exposed to natural challenge in the field, the majority are likely to become carriers of infection, even if there was previous sterile immunity. Thus, from the current work and other studies it appears that the carrier state is not deleterious to immunized cattle, nor a realistic threat to contact cattle, and furthermore may be unavoidable when cattle are exposed to field challenge. Immunity to ECF is dependent on the parasite becoming established in the host’s own cells (Brown et al., 1978; Eugui and Emery, 1981). Thus, in infection and treatment immunization, potentially virulent stabilate is used

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and the ensuing reaction is controlled with oxytetracycline. If the dose of stabilate is too low, infection may not be established and no immunity ensues. To avoid this possibility in the current work, relatively high doses of stabilate were used and some severe reactions occurred but all were successfully contained with Clexon or Halofuginone. Now that such drugs are available, the risk of overdosing with stabilate is greatly reduced and, in a field situation, is much less than the risk of underdosing and failing to immunize. Future work needs to be directed towards refining the immunization method particularly with reference to determining the optimum infective dose of stabilate in the infection and treatment schedule. The main emphasis of the current work was to determine whether theilerial stocks selected on the MAb test and cross-immunity trials could be successfully utilized to protect against field challenge. The fact that 100% protection was achieved using immunization with two selected stocks either in combination or singly clearly demonstrates that such an approach towards immunization can be made in T. p. paruu endemic areas. ACKNOWLEDGEMENTS

This work was conducted in collaboration with the Ministry of Agriculture and Livestock Development of Kenya. We are most grateful to Dr. S. Chema (Chief Veterinary Research Officer, Kenya) for his support in this work. Dr. J. Onyango (Provincial Director of Livestock Development, Coast Province), Dr. M. Kebaara (District Veterinary Officer, Kilifi), and their staff are acknowledged for assistance in the field. Messrs. C. Warui, S. Gichuki and J. Mureithi of the Coast Agriculture Research Station, Mtwapa, and S. Atudo of the ADC Farm, Kiswani are thanked for their help on the farms. The technical assistance of Messrs. J. Kiarie, R. Njamunggeh, T. Kiplangat, J. Mwenga, J. Nyika and M. Hamisi is acknowledged. Dr. G. Gettinby is thanked for assistance with statistical analysis. Pfizer Limited (Kenya) kindly provided Terramycin (LA) used in these studies. This is ILRAD Publication No. 418. REFERENCES Bailey, K.P., 1960. Notes on the rearing of Rhipicepholus uppendiculatus and their infection with Theileria parva for experimental transmission. Bull. Epizoot. Dis. Afr., 8: 33-43. Barnett, S.F., 1957. Theileriasis control. Bull. Epizoot. Dis. Afr., 5: 343-357. Brown, C.G.D., 1979. Propagation of Theileria. In: K. Maramorosch and H. Hirumi (Editors), Practical Tissue Culture Applications. Academic Press, NY, pp. 223-254. Brown, C.G.D., Crawford, J.G., Kanhai, G.K., Njuguna, L.M. and Stagg, D.A., 1978. Immunization of cattle against East Coast fever with lymphoblastoid cell lines infected and transformed by Theileria parua. In: J.K.H. Wilde (Editor), Tick-borne Diseases and their Vectors. University of Edinburgh, Edinburgh, pp. 331-333. Burridge, M.J., Morzaria, S.P., Cunningham, M.P. and Brown, C.G.D., 1972. Duration of immunity to East Coast fever (Theileria parua infection of cattle). Parasitology, 64: 511-515.

40 Cunningham, M.P., Brown, C.G.D., Burridge, M.J. and Purnell, R.E., 197 3. Cryopreservation of infective particles of Theileria pm-vu. Int. J. Parasitol., 3: 583-587. Dolan, T.T., 1981. Progress in the chemotherapy of theileriosis. In: A.D. Irvin, M.P. Cunningham and AS. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff, The Hague, pp. 186-208. Dolan, T.T., Radley, D.E., Brown, C.G.D., Cunningham, M.P., Morzaria, S.P. and Young, A.S., 1980. East Coast fever: further studies on the protection of cattle immunized with a combination of theilerial strains. Vet. Parasitol., 6: 325~-332. Eugui, E.M. and Emery, D.L., 1981. Genetically restricted cell-mediated cytotoxicity in cattle immune to Theileria porua. Nature, 290: 251.--253. Goddeeris, B.M., Katende, J.M., Irvin, A.D. and Chumo, R.S.C., 1982. Indirect fluorescent antibody test for experimental and epizootiological studies on East Coast fever (Theilerio parua infection of cattle). Evaluation of a cell culture schizont antigen fixed and stored in suspension. Res. Vet. Sci., 33: 360-365. Irvin, A.D., Dobbelaere, D.A.E., Mwamachi, D.M., Minami, T., Spooner, P.R. and Ocama, J.G.R., 1983. Immunization against East Coast fever: correlation between monoclonal antibody profiles of Theileria parua stocks and cross-immunity in vivo. Res. Vet. Sci., 35: 341-346. Irvin, A.D. and Morrison, W.I., 1985. Immunopathology, immunology and immunoprophylaxis of Theileria infections. In: E.J.L. Soulsby (Editor), Immunology, Immunoprophylaxis and Immunotherapy of Parasitic Infections. CRC Press Inc., Boca Raton, in press. Minami, T., Spooner, P.R., Irvin, A.D., Ocama, J.G.R., Dobbelaere, D.A.E. and Fujinaga, T., 1983. Characterization of stocks of Theileria parua by monoclonal antibody profiles. Res. Vet. Sci., 35: 334-340. Neitz, W.O., 1957. Theileriosis, gonderiosis and cytauxzoonosis: a review. Onderstepoort J. Vet. Res., 27: 275-430. Paling, R.W. and Geysen, D., 1981. Observations on Rwandan strains of Theileria parua and the value of T. parua Nyakizu as a possible vaccine strain. In: A.D. Irvin, M.P. Cunningham and A.S. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff, The Hague, pp. 238-241. Pinder, M. and Hewett, R.S., 1980. Monoclonal antibodies detect antigenic diversity in Theileria parua parasites. J. Immunol., 124: 1000-1001. Radley, D.E., 1978. Immunization against East Coast fever by chemoprophylaxis. FAO Technical Report 1. AG: DP/RAF/67/077 Rome. Radley, D.E., 1981. 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., Burridge, M.J., Cunningham, M.P., Kirimi, I.M., Purnell, R.E. and Young, A.S., 1975a. East Coast fever: 1. Chemoprophylactic immunization of cattle against Theileria parua (Muguga) and five theilerial strains. Vet. Parasitol., 1: 35-41. M.P., Musisi, Radley, D.E., Young, A.S., Brown, C.G.D., Burridge, M.J., Cunningham, F.L. and Purnell, R.E., 1975b. East Coast fever: 2. Cross-immunity trials with a Kenya strain of Theileria lawrencei. Vet. Parasitol., 1: 43-50. Radley, D.E., Newson, R.M., Cunningham, M.P. and Punyua, D.K., 1978. Effects of prolonged exposure of T. parua immunized cattle in an experimentally infected paddock. In: J.K.H. Wilde (Editor), Tick-borne Diseases and their Vectors. University of Edinburgh, Edinburgh, pp. 297-301. Robson, J., Pedersen, V., Odeke, G.M., Kamya, E.P. and Brown, C.G.D., 1977. East Coast fever immunization trials in Uganda: field exposure of Zebu cattle immunized with three isolates of Theileria parua. Trap. Anim. Health Prod., 9: 219-231. Survey of Kenya, 1972. Maps of Kenya 1:250,000 series Y503, Edition 3SK., Government of Kenya.

41 Uilenberg, G., Silayo, R.S., Mpangala, C., Tondeur, W., Tatchell, R.J. and Sanga, H.J.N., 1977. Studies on Theileriidae (Sporozoa) in Tanzania. X. A large scale field trial on immunization against cattle theileriosis. Tropenmed. Parasit., 28: 499-506. Winer, B.J., 1971. Statistical principles in experimental design. McGraw-Hill Inc., USA. pp. 261-305. Young, A.S., Brown, C.G.D., Burridge, M.J., Cunningham, M.P., Kirimi, 1.M. and Irvin, 1973. Observations on the cross-immunity between Theileria lawrencei A.D., (Serengeti) and Theileriaparua (Muguga) in cattle. Int. J. Parasitol., 3: 723-728. of infective stages of Young, A.S., Leitch, B.L. and Omwoyo, P.L., 1979. Induction Theileria parua by exposure of host ticks to high temperature. Vet. Rec., 105: 531~533. Young, A.S., Leitch, B.L. and Newson, R.M., 1981. The occurrence of a Theileriaparva carrier state in cattle from an East Coast fever endemic area of Kenya. In: A.D. Irvin, M.P. Cunningham and AS. Young (Editors), Advances in the Control of Theileriosis. Martinus Nijhoff, The Hague, pp. 60-62.