Veterinary Parasitology, 43 (1992) 15-24 Elsevier Science Publishers B.V., Amsterdam
15
Further evaluation of the use of buparvaquone in the infection and treatment method of immunizing cattle against Theileria parva derived from African buffalo (Syncerus caffer) P.N. Ngumi, A.S. Young, D. Lampard, S.K. Mining, S.G. Ndungu, A.C. Lesan, S . M . W i l l i a m s o n , A. L i n y o n y i a n d D . P . K a r i u k i
Protozoology Division, National Veterinary Research Centre, Muguga, Kenya Agricultural Research Institute, P.O. Box 32, Kikuyu, Kenya (Accepted 28 November 1991 )
ABSTRACT Ngumi, P.N., Young, A.S., Lampard, D., Mining, S.K., Ndungu, S.G., Lesan, A.C., Williamson, S.M., Linyonyi, A. and Kariuki, D.P., 1992. Further evaluation of the use ofbuparvaquone in the infection and treatment method of immunizing cattle against Theileria parva derived from African buffalo (Syncerus caffer). Vet. Parasitol, 43:15-24. Three experiments were undertaken to determine the efficacy of different doses of buparvaquone in the infection and treatment immunization of cattle against Theileria parva derived from African buffalo (Syncerus caffer). Two of these experiments also compared buparvaquone with standard doses of long- and short-acting formulations of oxytetracycline. In addition, different dilutions of stabilates were used in the experiments. In the first experiment, a 10 - L0 dilution of stabilate was used to infect groups of cattle treated with buparvaquone at doses of between 5 and 0.625 mg kg-~ body weight (bwt) on Day 0 after infection. All control cattle developed severe theileriosis and none of the treatment regimes (including those utilizing long-acting oxytetracycline) prevented the development of theileriosis. Treatment with buparvaquone at 2.5 mg kg- ~bwt or oxytetracycline gave the most satisfactory results. In the second experiment when the sporozoite dose was reduced to 10 -2.o dilution, buparvaquone treatment at 5 and 2.5 mg kg-I bwt and short- and long-acting formulations of oxytetracycline reduced reactions greatly. While all the oxytetracycline treated animals produced a serological response and were immune to a 50-fold higher challenge with the immunizing stabilate, several animals in the buparvaquone groups did not show a serological response and were not immune to challenge. In the third experiment, groups of cattle were infected with 10-L2, 10-L4 and 10-1.6 dilutions of stabilate and were treated with 2.5 mg kg- ~bwt ofbuparvaquone. No animals developed severe theileriosis and all seroconverted. On homologous challenge, however, two out of 14 cattle showed severe reactions. It was concluded that further work on immunization using buparvaquone treatment at 2.5 mg kg bwl and 10-1.6 dilution of the stabilate would have to be carried out before such a system could be used in the field.
Correspondence to." A.S. Young, International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
16
P.N. NGUMI ET AL.
INTRODUCTION
A major problem in many areas of Kenya and elsewhere in East Africa is the control of Theileria parva infection in cattle, when the parasite is derived from African buffalo (Syncerus caffer). This parasite, transmitted by the ixodid tick Rhipicephalus appendiculatus, had originally been described by Neitz (1955) as Theileria lawrencei, later as T. parva lawrencei (Uilenberg, 1976) and it now has been r e c o m m e n d e d that it should be referred to as T. parva (Anonymous, 1989). Buparvaquone (Butalex ®, Pitman-Moore, Berkhamstead, U K ) , a hydroxynaphthoquinone has been shown to a more effective therapeutic drug for treating theileriosis than parvaquone (Clexon ®, PitmanMoore, Berkhamstead, U K ) (McHardy et al., 1985). In previous experiments it has been shown that buparvaquone has potential for use in the infection and treatment m e t h o d of immunization of cattle against T. parva infection (McHardy and Wekesa, 1985; Mutugi et al., 1988a, 1991 ). Mutugi et al. (1988a, 1991 ) reported that it appeared to be particularly useful in the infection and treatment method using T. parva stabilates derived from African buffalo. The use of these tick stabilates at a dose which protects against homologous challenge resulted in breakthroughs in the chemoprophylaxis provided by oxytetracycline in the standard infection and treatment method (Mutugi et al., 1988b). In the present study, the use of buparvaquone at different doses in the infection and treatment m e t h o d using five different dilutions of a sporozoite stabilate of T. parva derived from African buffalo was compared with the use of oxytetracycline treatments. MATERIALS AND METHODS
Cattle Cattle used in experiments were high grade Friesian/Hereford crosses over 1 year of age: they gave a negative reaction in the indirect fluorescent antibody test (IFAT) using T. parva schizont antigen (Burridge and Kimber, 1972). Theilerial parasites The theilerial parasite used was T. parva (O1 Pejeta buffalo stock). It was derived by feeding ticks on buffalo 7014 captured as a calf on O1 Pejeta Ranch, Nanyuki, Rift Valley Province, Kenya (Mutugi et al., 1988a). The sporozoite stabilate was designated IL3081. It was used in experiments at either 10°, 10- l, 10- 1.3 10-- 1.6 10 - 1.8 of 10- 2 dilutions using Eagle's minimal essential m e d i u m (Gibco, BLR, Paisley, U K ) with 3.5% bovine plasma albumin and
BUPARVAQUONE IN IMMUNIZATION OF CATTLE AGAINST T. PARVA
17
7.5% glycerol as diluent. One milliliter of undiluted or diluted stabilate was inoculated subcutaneously into the cheek unless specified otherwise.
Drugs Buparvaquone (BW720C; 2-hydroxy-3- (trans-4-butyl-cyclohexyl methyl1, 4-naphthoquinone) was used for treatment (McHardy, 1989). It was formulated at 50 mg ml-~ (Butalex, Pitman-Moore) and given at the required dose, according to body weight (bwt) by intramuscular injection into the gluteal muscle. For comparative studies, a long-acting formulation ofoxytetracycline (Terramycin LA, Pfizer Ltd., Sandwich, U K ) formulated at 200 mg ml-~ and a short-acting formulation of oxytetracycline (Medamycin 100, TechAmerica Co. Inc., Elwood, KS, USA) formulated as a 100 mg m l - ~solution were given at the required dosage by intramuscular injection into the gluteal muscles.
Experimental design Three experiments were carried out using five different dilutions of T. parva stabilate and different doses of buparvaquone compared with standard doses of oxytetracycline.
Experiment 1 Thirty-five cattle were divided into seven groups of five animals. They were inoculated with a 10-~ dilution of stabilate IL3081 and the groups were treated as follows. Group 1: buparvaquone 5.0 mg kg- 1 bwt on Day 0 after infection. Group 2: buparvaquone 2.5 mg kg-1 bwt on Day 0 after infection. Group 3: buparvaquone 1.25 mg kg- ~bwt on Day 0 after infection. Group 4: buparvaquone 0.625 mg kg- ~bwt on Day 0 after infection. Group 5: long-acting oxytetracycline treatment 20 mg kg-1 bwt on Day 0 after infection. Group 6: long-acting oxytetracycline treatment 20 mg kg-~ bwt on Day 0 and 4 after infection. Group 7: no treatment.
Experiment 2 Thirty cattle were divided into groups of five cattle. They were inoculated with 1 ml of a 10 -2 dilution of the stabilate and were then treated as follows. Group 1: buparvaquone 5 mg kg- 1 bwt on Day 0 after infection. Group 2: buparvaquone 2.5 mg kg- 1 bwt on Day 0 after infection. Group 3: buparvaquone 1.25 mg kg-1 bwt on Day 0 after infection.
I8
P.N. NGUMI ET AL.
Group 4: long-acting oxytetracycline 20 mg kg-~ bwt on Day 0 after infection. Group 5: short-acting oxytetracycline 10 mg kg-l bwt on Day 0 and 4 after infection. Group 6: no treatment.
Experiment 3 Fifteen cattle were divided into three groups of five cattle. The cattle were treated as follows. Group 1:1 ml ofstabilate at 10 13 dilution followed by buparvaquone 2.5 mg kg- 1 bwt on Day 0. Group 2:1 ml of stabilate at 10-16 dilution followed by buparvaquone 2.5 mg kg- 1 btw on Day 0. Group 3:1 ml of stabilate at 10-~.8 dilution followed by buparvaquone 2.5 mg k g - 1 bwt on Day 0.
Monitoring cattle for theilerial infection After inoculation of stabilate, rectal temperatures were taken daily. Needle biopsies were taken daily from the left paratoid lymph node from Day 5 after inoculation of stabilate. Lymph node biopsy smears were stained in Giemsa and examined for the presence of schizonts. When the local drainage lymph nodes became positive or from Day 10 after inoculation, needle biopsies were also taken from the contralateral prescapular lymph nodes. Lymph node biopsies were obtained at daily intervals until the animals died or if no schizonts or clinical symptoms were seen for several days. Peripheral blood smears were prepared at daily intervals from Day 10 after inoculation until the animals recovered or died. Cattle were bled for serology on Days 28, 35 and 42 after infection and the serum assayed in the IFAT to detect antibody response against T. parva. A febrile response was defined as a rectal temperature of 39.5 °C or above associated with theilerial parasitosis. Recovery was defined as the day of disappearance of theilerial schizonts from lymph node biopsies. In Experiment 1, surviving cattle plus two susceptible steers were challenged with 0.5 ml of undiluted St.IL3081, 45 days after inoculation. In Experiment 2, the cattle were challenged with 0.5 ml of undiluted St.IL3081 on Day 110 after inoculation. In Experiment 3, the cattle were challenged with 0.5 ml of undiluted St.IL3081 on Day 35 after inoculation. The cattle were monitored for the development of theilerial infection. RESULTS
The results of Experiment 1 are shown in Table 1. Clinical reactions occurred in all groups as did deaths due to theileriosis. All five control cattle
BUPARVAQUONEIN IMMUNIZATIONOF CATTLEAGAINST72 PAR VA
19
TABLE 1 The efficacy of different doses of buparvaquone and a long-acting formulation of oxytetracycline in infection and treatment immunization using 10-J.o dilution of Theileria parva sporozoite stabilate IL3081 Group Treatment no.
1
2
3
4
5
6
7
Buparvaquone ( 5 mg kg- i bwt ) Total days of: Buparvaquone (2.5 mg kg -~ bwt) Total days of: Buparvaquone (1.25 mg kg -1 bwt) Total days of: Buparvaquone (0.625 mgkg -1 bwt) Total days of: Terramycin LA (Day 0, 20 mg kg -1 bwt ) Total days of: TerramycinLA (Days 0 and 4, 20 mg kg- J bwt ) Total days of: No treatment Total days of:
Mean days to: Schizont Fever
Recovery Death
Positive Immune serology to in theIFAT challenge
19.0(4)
23.0(2)
20.0(3)
21.0(1)
3/4
3/41
6 12.6(5)
4 19.0(4)
24.7(3)
22.0(2)
3/3
3/3
31 8.8(5)
7 14.8(5)
21(1)
17.0(4)
1/1
1/1
44 7.4(5)
24 12.5(4)
27(1)
15.8(4)
1/1
1/1
44 10.6(5)
13 16.6(5)
24.0(4)
21(1)
4/4
4/4
44 10.0(5)
19 15.0(2)
19.7(3)
20.0(2)
3/3
3/3
34 7.6(5) 45
10 11.8(5) 17
16.4(5)
-
-
-
~One animal did not react to initial infection. -, No reaction. Figures in parentheses are the number out of five undergoing a particular reaction.
died compared with one in the 5.0 mg kg-1 bwt, two in the 2.5 mg kg-l bwt, four in the 1.25 mg k g - l bwt, four in the 0.625 mg kg-l bwt buparvaquone treatment groups and one and two in the Terramycin LA treatment groups (groups 5 and 6, respectively). If the total days of patent theilerial schizonts and fever are compared between the groups, it can be seen that the doses of buparvaquone above 1.25 mg kg-~ bwt reduced the days of patent schizonts and fever dramatically. Terramycin LA treatment failed to reduce the days of patent schizonts or fever greatly but recovery rates were higher. The serological conversion was good in all groups except in the 5 mg buparvaquone kg-l bwt group where one animal remained negative and proved to be susceptible on challenge (Table 1 ). All other surviving cattle were i m m u n e (Table 1 ).
20
P.N. NGUMI ET AL.
In Experiment 2, no theilerial reactions were seen in any of the buparvaquone treated groups except in the 1.25 mg kg- ~ bwt group which showed a similar theilerial reaction to the control untreated group with two out of five cattle dying in both groups (Table 2). The oxytetracycline treated groups showed no or minimal theilerial reaction. Major differences were observed between the groups in the serological conversion and immunity to challenge. None of the five animals in the 5 mg kg-1 buparvaquone group showed a serological response and none were immune while out of five animals in the 2.5 mg buparvaquone group four showed a serological response and four were immune (Table 2 ). Even in the 1.25 mg buparvaquone group where three out of three showed a serological response, only two proved to be immune (Table 2). This compares poorly with seroconversion of five out of five in the Terramycin or Medamycin treated groups where all the animals proved to be immune (Table 2). The surviving control cattle all proved to be serologic converters and immune on challenge. In Experiment 3, mild reactions were seen in all groups receiving either 10 -13, 10 -16 or 10 -18 dilutions of ST IL3081 and buparvaquone at 2.5 mg TABLE2
The efficacy of different doses of buparvaquone and long- and short-acting formulations of oxytetracyclines in infection and treatment immunization using 10 -2.o dilution of Theileria parva sporozoite stabilate IL3081 Group Treatment no.
Mean days to: Schizont Fever
1
2
Buparvaquone (5.0 mgkg -~ bwt)
.
Total days of:
0
Buparvaquone (1.25 mgkg -1 bwt)
Total days of: 4
TerramycinLA ( 20 mg kg- 1bwt )
Total days of: 5
Medamycin 100 (10 mgkg-~ b w t × 2 )
Total days of: 6
No treatment
Total days of:
. 0 -
Buparvaquone (2.5 mgkg -~ bwt)
Total days of: 3
.
Recovery Death .
-
0 13.0(4)
0 18.0(3)
19.0(2)
23 16.0(2)
9 23(1)
22.5(2)
7 . 0 11.6(5) 25
-
25.0(2)
-
Positive Immune serology to in theIFAT challenge 0/5
0/5
4/5
4/5
3/3
2/3
5/5
5/5
5/5
5/5
3/3
3/3
1 .
. 0 14.4(5) 12
.
20.3(3)
20.5(2)
-, No reaction.
Figures in parentheses are the number out of five undergoing a particular reaction.
BUPARVAQUONEIN IMMUNIZATIONOF CATTLEAGAINST ~CPARVA
21
TABLE 3 The efficacy of different doses of buparvaquone infection and treatment immunization using 10- L3, 10-1.6 and 10- L8 dilution of Theileria parva sporozoite stabilate IL3081 Group Treatment no.
1
2
3
Buparvaquone ( 2.5 mg kg- 1 bwt, 10-L3 dilution of stabilate) Total days of: Buparvaquone (2.5 mg kg- 1bwt, 10-16 dilution of stabilate) Total days of: Buparvaquone ( 1.25 mg kg- 1bwt, 10- ~.8 dilution of stabilate) Total days of:
Mean days to: Schizont Fever
Recovery Death
Positive Immune serology to in thelFAT challenge
26.0(2)
-
30.5(2)
-
5/5
4/5
8 22.0(3)
0 -
25.0(3)
-
5/5
4/5
8 37( 1 )
0 -
38 ( 1 )
-
5/5
4/41
1
0
~One animal died of other causes. Figures in parentheses are the number out of five undergoing a particular reaction.
kg- 1 bwt (Table 3 ). All cattle seroconverted by Day 28 and on challenge with undiluted stabilate on Day 35, 12 out of the 14 cattle challenged were immune. DISCUSSION
The infection and treatment method of immunization depends on the use of a stabilate of wide immunogenicity containing a number of different immunogenic parasite populations infecting a large proportion of animals to be immunized (preferably 100%) while the use of chemoprophylaxis aims to prevent the development of clinical disease so that the animals become safely i m m u n e to unlimited homologous challenge (Mutugi et al., 1988b, 1989 ). It is possible to overcome the effect of the oxytetracycline treatment as it is not schizonticidal but depends on slowing down the development of the theilerial infection so that the i m m u n e response of the cow can control the infection (Spooner, 1990). Usually a balance can be obtained between the doses of sporozoite and oxytetracycline but when large numbers of animals are used, there are exceptions. This makes infection and treatment difficult to monitor in the field. Both parvaquone and buparvaquone (Clexon and Butalex, respectively Pitman-Moore) can be used in infection and treatment immunization. Par-
22
P.N. N G U M I ET AL.
vaquone, if used on Days 0 or 4 after infection at its therapeutic level (20 mg kg- bwt), can block the development of parasites so that cattle do not become immune. Therefore, it has to be used on Day 8 after infection or later (Dolan et al., 1984, 1988; Young et al., 1990). On the other hand, buparvaquone treatment given on Day 0 after infection at its reported therapeutic level (2.5 mg kg-1 bwt) has been reported to allow the development of immunity (McHardy and Wekesa, 1985; Mutugi et al., 1988b, 1991 ). Hence it was believed that buparvaquone treatment would be safer than oxytetracycline treatment (Mutugi et al., 1988a) as it had schizonticidal effects when given on Day 0 but would allow the development of immunity (McHardy and Wekesa, 1985; Mutugi et al., 1988a). Therefore it was hoped that buparvaquone would prevent clinical reactions which can develop when T. parva derived from buffalo is used in immunization with oxytetracycline treatment. Unfortunately, in these experiments, where the drug was titrated against two levels of sporozoite stabilate dose of T. parva, the results were not as anticipated. When the higher dose of T. parva stabilate was used ( 10 ~ dilution), low doses of buparvaquone (2.5 and 1.25 mg kg -1 bwt) did not prevent the clinical disease as compared with the control cattle and a high proportion died. Although 2.5 mg kg-~ ofbuparvaquone did reduce the theilerial reaction, two animals still died. Buparvaquone treatment at 5 mg kg considerably reduced the clinical reaction but one animal died of theileriosis and one did not become immune. Hence there would appear to be no advantages ofbuparvaquone over oxytetracycline when used with this stabilate dose. When the stabilate dose was reduced ten-fold in Experiment 2, reactions in all groups were reduced and only two of five animals in the control group died. Hence the infecting dose appeared to be at the level of LDs0 which we have already shown with oxytetracycline treatment to be a usually effective and safe immunogenic dose of stabilate (Mutugi et al., 1988b, 1989). This was confirmed in this experiment where none of the oxytetracycline-treated animals showed theilerial reaction during immunization and all were immune to challenge with a 50-fold higher dose of homologous stabilate. The buparvaquone-treated animals showed a different pattern in the 5 mg kg-l group where none reacted, no serological titer developed and all were susceptible on challenge. It appears that this dose of drug blocked the development of infection and thus immunity with this dose of stabilate. Again in the 2.5 mg kg- 1 group, clinical reactions were prevented but one out of five animals was not immune. The 1.25 mg kg-1 dose of buparvaquone did not prevent the development of clinical reactions and one out of three surviving cattle was not immune, It was of interest that even when the stabilate dose was reduced, 1.25 mg kg- ~ bwt of buparvaquone was not effective in preventing development of clinical theileriosis. When intermediate doses of sporozoites were investigated in Experiment 3, the clinical reaction of cattle receiving 2.5 mg kg- 1 bwt of buparvaquone
BUPARVAQUONE IN IMMUNIZATION OF CATTLE AGAINST T 1~4R1~4
23
was reduced and immunity to challenge appeared to be improved, particularly with the 10-~.6 dilution of stabilate. No control cattle were used in Experiment 3 as extensive data were available from previous experiments using dilutions of 10°, 10 -1 and 10 -2 (Mutugi et al., 1988a,b). It can be concluded that using this stabilate it might be more desirable to use oxytetracyclines than buparvaquone as the therapeutic level to assure the infection of parasites with the development of homologous immunity without clinical reaction using buparvaquone treatment might be too narrow. This principle might not apply to other T. parva stabilates as indicated by McHardy and Wekesa ( 1985 ) and Mutugi et al. (1988a). The reasons for the choice of buffalo stock of T. parva for these experiments were that it provided protection against Narok District stocks of T. parva derived from buffalo in a geographically distant area (Mutugi et al., 1988b ) and because a previous stabilate prepared from this buffalo had worked well in the infection and treatment immunization at 10 -2.o dilution using either buparvaquone or oxytetracycline (Mutugi et al., 1988a,b). In the present experiments, however, neither 10 -l-° nor 10 -2.o dilutions of stabilate IL3081 worked well with buparvaquone treatment. Intermediate dilutions of stabilate appeared to work better and 10-16 dilution may be suitable for field immunization. To broaden these observations, more extensive experiments need to be undertaken. Meanwhile, treatment with either long- or short-acting formulations of oxytetracycline, for infection and treatment immunization against buffalo-derived T. parva infection with 10 -2° dilution of ST. IL3081 is recommended. ACKNOWLEDGMENTS We would like to thank Pitman-Moore, Europe for the supply of buparvaquone and their advice in experiments particularly, Dr. N. McHardy and Dr. D. Morgan. They also provided some financial support. This paper was published by kind permission of the Director of the National Veterinary Research Centre, Muguga. During these studies, Dr. A.S. Young and D. Lampard were employed by the UK Overseas Development Administration.
REFERENCES Anonymous, ! 989. Nomenclaturein Theileria. In: T.T. Dolan (Editor), Theileriosisin Eastern, Central and Southern Africa. International Laboratoryfor Research on Animal Diseases, Nairobi, pp. 182-186. Burridge, M.J. and Kimber,C.D., 1972. The indirectfluorescentantibodytest for experimental
24
P.N. N G U M I ET AL.
East Coast fever (Theileria parva infection of cattle). Evaluation of a cell culture antigen. Res. Vet. Sci., 13: 451-455. Dolan, T.T., Linyonyi, A., Mbogo, S.K. and Young, A.S., 1984. A comparison of long-acting oxytetracycline and parvaquone for immunization against East Coast fever by infection and treatment. Res. Vet. Sci., 37:175-178. Dolan, T.T., Linyonyi, A., McHardy, N., Bond, A.L. and Clampitt, R.D., 1988. Chemotherapy of East Coast fever: parvaquone treatment of Theileria parva parva at intervals after infection. Res. Vet. Sci., 44:15-20. McHardy, N., 1989. Buparvaquone, the new antitheilerial: a review of its efficacy and safety. In: T.T. Dolan (Editor), Theileriosis in Eastern, Central and Southern Africa. International Laboratory Research for Animal Disease, Nairobi, pp. 158-165. McHardy, N. and Wekesa, L.S., 1985. Buparvaquone (BW720C) - - a new anti-theilerial naphthoquinone: its role in therapy and prophylaxis. In: A.D. lrvin (Editor), Immunization against Theileriosis in Africa. International Laboratory for Research on Animal Diseases, Nairobi, p.88. McHardy, N., Wekesa, L.S., Hudson, A.T. and Randal, A.W., 1985. Anti-theilerial activity of BW720C (buparvaquone): a comparison with parvaquone. Res. Vet. Sci., 39: 29-33. Mutugi, J.J., Young, A.S., Maritim, A.C., Linyonyi, A., Mbogo, S.K. and Leitch, B.L. 1988a. Immunization using varying infective doses of Theileria parva lawrencei sporozoites derived from an African buffalo (S.vncerus caller) and treatment with buparvaquone. Parasitology, 96: 391-401. Mutugi, J.J., Young, A.S., Maritim, A.C., Ndungu, S.G., Stagg, D.A., Grootenhuis, J.G. and Leitch, B.L., 1988b. Immunization of cattle against theileriosis using varying doses of Theileria parva parva sporozoites and oxytetracycline treatments. Int. J. Parasitol., 18:453-461. Mutugi, J.J., Young, A.S., Maritim, A.C., Ndungu, S.G., Mining, S.K., Linyonyi, A., Ngumi, P.N., Leitch, B.L., Morzaria, S.P. and Dolan, T.T., 1989. Immunization of cattle against theileriosis in Coast Province of Kenya: Laboratory evaluation of a large Theileria parva parva stabilate for use in "Infection and treatment" immunization in the field. Res. Vet. Sci., 47: 170-179. Mutugi J.J., Young, A.S., Kariuki, D.P., Morzaria, S.P. and Ole Tameno, J.M., 1991. Epidemiological observations on theileriosis following field immunization using infection and treatment with either oxytetracycline or buparvaquone. Trop. Anita. Health Prod., 23: 7581. Neitz, W.O., 1955. Corridor Disease, a fatal form of bovine theileriosis encountered in Zululand. Bull. Epizoot. Dis. Afr., 3: 121-123. Spooner, P.R., 1990. The effects of oxytetracycline on Theileria parva in vitro. Parasitology, 100:11-17.
Uilenberg, G., 1976. Tick-borne diseases and their vectors. 2. Epizootiology of tick-borne disease. World Anita. Rev., 17: 8-15. Young, A.S., Leitch, B.L., Dolan, T.T., Mbogo, S.K., Ndungu, S.G., Grootenhuis, J.G. and de Castro, J.J., 1990. Evaluation of infection and treatment methods in immunization of improved cattle against theileriosis in an endemic area of Kenya. Vet. Parasitol., 35: 239-257.
15
Further evaluation of the use of buparvaquone in the infection and treatment method of immunizing cattle against Theileria parva derived from African buffalo (Syncerus caffer) P.N. Ngumi, A.S. Young, D. Lampard, S.K. Mining, S.G. Ndungu, A.C. Lesan, S . M . W i l l i a m s o n , A. L i n y o n y i a n d D . P . K a r i u k i
Protozoology Division, National Veterinary Research Centre, Muguga, Kenya Agricultural Research Institute, P.O. Box 32, Kikuyu, Kenya (Accepted 28 November 1991 )
ABSTRACT Ngumi, P.N., Young, A.S., Lampard, D., Mining, S.K., Ndungu, S.G., Lesan, A.C., Williamson, S.M., Linyonyi, A. and Kariuki, D.P., 1992. Further evaluation of the use ofbuparvaquone in the infection and treatment method of immunizing cattle against Theileria parva derived from African buffalo (Syncerus caffer). Vet. Parasitol, 43:15-24. Three experiments were undertaken to determine the efficacy of different doses of buparvaquone in the infection and treatment immunization of cattle against Theileria parva derived from African buffalo (Syncerus caffer). Two of these experiments also compared buparvaquone with standard doses of long- and short-acting formulations of oxytetracycline. In addition, different dilutions of stabilates were used in the experiments. In the first experiment, a 10 - L0 dilution of stabilate was used to infect groups of cattle treated with buparvaquone at doses of between 5 and 0.625 mg kg-~ body weight (bwt) on Day 0 after infection. All control cattle developed severe theileriosis and none of the treatment regimes (including those utilizing long-acting oxytetracycline) prevented the development of theileriosis. Treatment with buparvaquone at 2.5 mg kg- ~bwt or oxytetracycline gave the most satisfactory results. In the second experiment when the sporozoite dose was reduced to 10 -2.o dilution, buparvaquone treatment at 5 and 2.5 mg kg-I bwt and short- and long-acting formulations of oxytetracycline reduced reactions greatly. While all the oxytetracycline treated animals produced a serological response and were immune to a 50-fold higher challenge with the immunizing stabilate, several animals in the buparvaquone groups did not show a serological response and were not immune to challenge. In the third experiment, groups of cattle were infected with 10-L2, 10-L4 and 10-1.6 dilutions of stabilate and were treated with 2.5 mg kg- ~bwt ofbuparvaquone. No animals developed severe theileriosis and all seroconverted. On homologous challenge, however, two out of 14 cattle showed severe reactions. It was concluded that further work on immunization using buparvaquone treatment at 2.5 mg kg bwl and 10-1.6 dilution of the stabilate would have to be carried out before such a system could be used in the field.
Correspondence to." A.S. Young, International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
16
P.N. NGUMI ET AL.
INTRODUCTION
A major problem in many areas of Kenya and elsewhere in East Africa is the control of Theileria parva infection in cattle, when the parasite is derived from African buffalo (Syncerus caffer). This parasite, transmitted by the ixodid tick Rhipicephalus appendiculatus, had originally been described by Neitz (1955) as Theileria lawrencei, later as T. parva lawrencei (Uilenberg, 1976) and it now has been r e c o m m e n d e d that it should be referred to as T. parva (Anonymous, 1989). Buparvaquone (Butalex ®, Pitman-Moore, Berkhamstead, U K ) , a hydroxynaphthoquinone has been shown to a more effective therapeutic drug for treating theileriosis than parvaquone (Clexon ®, PitmanMoore, Berkhamstead, U K ) (McHardy et al., 1985). In previous experiments it has been shown that buparvaquone has potential for use in the infection and treatment m e t h o d of immunization of cattle against T. parva infection (McHardy and Wekesa, 1985; Mutugi et al., 1988a, 1991 ). Mutugi et al. (1988a, 1991 ) reported that it appeared to be particularly useful in the infection and treatment method using T. parva stabilates derived from African buffalo. The use of these tick stabilates at a dose which protects against homologous challenge resulted in breakthroughs in the chemoprophylaxis provided by oxytetracycline in the standard infection and treatment method (Mutugi et al., 1988b). In the present study, the use of buparvaquone at different doses in the infection and treatment m e t h o d using five different dilutions of a sporozoite stabilate of T. parva derived from African buffalo was compared with the use of oxytetracycline treatments. MATERIALS AND METHODS
Cattle Cattle used in experiments were high grade Friesian/Hereford crosses over 1 year of age: they gave a negative reaction in the indirect fluorescent antibody test (IFAT) using T. parva schizont antigen (Burridge and Kimber, 1972). Theilerial parasites The theilerial parasite used was T. parva (O1 Pejeta buffalo stock). It was derived by feeding ticks on buffalo 7014 captured as a calf on O1 Pejeta Ranch, Nanyuki, Rift Valley Province, Kenya (Mutugi et al., 1988a). The sporozoite stabilate was designated IL3081. It was used in experiments at either 10°, 10- l, 10- 1.3 10-- 1.6 10 - 1.8 of 10- 2 dilutions using Eagle's minimal essential m e d i u m (Gibco, BLR, Paisley, U K ) with 3.5% bovine plasma albumin and
BUPARVAQUONE IN IMMUNIZATION OF CATTLE AGAINST T. PARVA
17
7.5% glycerol as diluent. One milliliter of undiluted or diluted stabilate was inoculated subcutaneously into the cheek unless specified otherwise.
Drugs Buparvaquone (BW720C; 2-hydroxy-3- (trans-4-butyl-cyclohexyl methyl1, 4-naphthoquinone) was used for treatment (McHardy, 1989). It was formulated at 50 mg ml-~ (Butalex, Pitman-Moore) and given at the required dose, according to body weight (bwt) by intramuscular injection into the gluteal muscle. For comparative studies, a long-acting formulation ofoxytetracycline (Terramycin LA, Pfizer Ltd., Sandwich, U K ) formulated at 200 mg ml-~ and a short-acting formulation of oxytetracycline (Medamycin 100, TechAmerica Co. Inc., Elwood, KS, USA) formulated as a 100 mg m l - ~solution were given at the required dosage by intramuscular injection into the gluteal muscles.
Experimental design Three experiments were carried out using five different dilutions of T. parva stabilate and different doses of buparvaquone compared with standard doses of oxytetracycline.
Experiment 1 Thirty-five cattle were divided into seven groups of five animals. They were inoculated with a 10-~ dilution of stabilate IL3081 and the groups were treated as follows. Group 1: buparvaquone 5.0 mg kg- 1 bwt on Day 0 after infection. Group 2: buparvaquone 2.5 mg kg-1 bwt on Day 0 after infection. Group 3: buparvaquone 1.25 mg kg- ~bwt on Day 0 after infection. Group 4: buparvaquone 0.625 mg kg- ~bwt on Day 0 after infection. Group 5: long-acting oxytetracycline treatment 20 mg kg-1 bwt on Day 0 after infection. Group 6: long-acting oxytetracycline treatment 20 mg kg-~ bwt on Day 0 and 4 after infection. Group 7: no treatment.
Experiment 2 Thirty cattle were divided into groups of five cattle. They were inoculated with 1 ml of a 10 -2 dilution of the stabilate and were then treated as follows. Group 1: buparvaquone 5 mg kg- 1 bwt on Day 0 after infection. Group 2: buparvaquone 2.5 mg kg- 1 bwt on Day 0 after infection. Group 3: buparvaquone 1.25 mg kg-1 bwt on Day 0 after infection.
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P.N. NGUMI ET AL.
Group 4: long-acting oxytetracycline 20 mg kg-~ bwt on Day 0 after infection. Group 5: short-acting oxytetracycline 10 mg kg-l bwt on Day 0 and 4 after infection. Group 6: no treatment.
Experiment 3 Fifteen cattle were divided into three groups of five cattle. The cattle were treated as follows. Group 1:1 ml ofstabilate at 10 13 dilution followed by buparvaquone 2.5 mg kg- 1 bwt on Day 0. Group 2:1 ml of stabilate at 10-16 dilution followed by buparvaquone 2.5 mg kg- 1 btw on Day 0. Group 3:1 ml of stabilate at 10-~.8 dilution followed by buparvaquone 2.5 mg k g - 1 bwt on Day 0.
Monitoring cattle for theilerial infection After inoculation of stabilate, rectal temperatures were taken daily. Needle biopsies were taken daily from the left paratoid lymph node from Day 5 after inoculation of stabilate. Lymph node biopsy smears were stained in Giemsa and examined for the presence of schizonts. When the local drainage lymph nodes became positive or from Day 10 after inoculation, needle biopsies were also taken from the contralateral prescapular lymph nodes. Lymph node biopsies were obtained at daily intervals until the animals died or if no schizonts or clinical symptoms were seen for several days. Peripheral blood smears were prepared at daily intervals from Day 10 after inoculation until the animals recovered or died. Cattle were bled for serology on Days 28, 35 and 42 after infection and the serum assayed in the IFAT to detect antibody response against T. parva. A febrile response was defined as a rectal temperature of 39.5 °C or above associated with theilerial parasitosis. Recovery was defined as the day of disappearance of theilerial schizonts from lymph node biopsies. In Experiment 1, surviving cattle plus two susceptible steers were challenged with 0.5 ml of undiluted St.IL3081, 45 days after inoculation. In Experiment 2, the cattle were challenged with 0.5 ml of undiluted St.IL3081 on Day 110 after inoculation. In Experiment 3, the cattle were challenged with 0.5 ml of undiluted St.IL3081 on Day 35 after inoculation. The cattle were monitored for the development of theilerial infection. RESULTS
The results of Experiment 1 are shown in Table 1. Clinical reactions occurred in all groups as did deaths due to theileriosis. All five control cattle
BUPARVAQUONEIN IMMUNIZATIONOF CATTLEAGAINST72 PAR VA
19
TABLE 1 The efficacy of different doses of buparvaquone and a long-acting formulation of oxytetracycline in infection and treatment immunization using 10-J.o dilution of Theileria parva sporozoite stabilate IL3081 Group Treatment no.
1
2
3
4
5
6
7
Buparvaquone ( 5 mg kg- i bwt ) Total days of: Buparvaquone (2.5 mg kg -~ bwt) Total days of: Buparvaquone (1.25 mg kg -1 bwt) Total days of: Buparvaquone (0.625 mgkg -1 bwt) Total days of: Terramycin LA (Day 0, 20 mg kg -1 bwt ) Total days of: TerramycinLA (Days 0 and 4, 20 mg kg- J bwt ) Total days of: No treatment Total days of:
Mean days to: Schizont Fever
Recovery Death
Positive Immune serology to in theIFAT challenge
19.0(4)
23.0(2)
20.0(3)
21.0(1)
3/4
3/41
6 12.6(5)
4 19.0(4)
24.7(3)
22.0(2)
3/3
3/3
31 8.8(5)
7 14.8(5)
21(1)
17.0(4)
1/1
1/1
44 7.4(5)
24 12.5(4)
27(1)
15.8(4)
1/1
1/1
44 10.6(5)
13 16.6(5)
24.0(4)
21(1)
4/4
4/4
44 10.0(5)
19 15.0(2)
19.7(3)
20.0(2)
3/3
3/3
34 7.6(5) 45
10 11.8(5) 17
16.4(5)
-
-
-
~One animal did not react to initial infection. -, No reaction. Figures in parentheses are the number out of five undergoing a particular reaction.
died compared with one in the 5.0 mg kg-1 bwt, two in the 2.5 mg kg-l bwt, four in the 1.25 mg k g - l bwt, four in the 0.625 mg kg-l bwt buparvaquone treatment groups and one and two in the Terramycin LA treatment groups (groups 5 and 6, respectively). If the total days of patent theilerial schizonts and fever are compared between the groups, it can be seen that the doses of buparvaquone above 1.25 mg kg-~ bwt reduced the days of patent schizonts and fever dramatically. Terramycin LA treatment failed to reduce the days of patent schizonts or fever greatly but recovery rates were higher. The serological conversion was good in all groups except in the 5 mg buparvaquone kg-l bwt group where one animal remained negative and proved to be susceptible on challenge (Table 1 ). All other surviving cattle were i m m u n e (Table 1 ).
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P.N. NGUMI ET AL.
In Experiment 2, no theilerial reactions were seen in any of the buparvaquone treated groups except in the 1.25 mg kg- ~ bwt group which showed a similar theilerial reaction to the control untreated group with two out of five cattle dying in both groups (Table 2). The oxytetracycline treated groups showed no or minimal theilerial reaction. Major differences were observed between the groups in the serological conversion and immunity to challenge. None of the five animals in the 5 mg kg-1 buparvaquone group showed a serological response and none were immune while out of five animals in the 2.5 mg buparvaquone group four showed a serological response and four were immune (Table 2 ). Even in the 1.25 mg buparvaquone group where three out of three showed a serological response, only two proved to be immune (Table 2). This compares poorly with seroconversion of five out of five in the Terramycin or Medamycin treated groups where all the animals proved to be immune (Table 2). The surviving control cattle all proved to be serologic converters and immune on challenge. In Experiment 3, mild reactions were seen in all groups receiving either 10 -13, 10 -16 or 10 -18 dilutions of ST IL3081 and buparvaquone at 2.5 mg TABLE2
The efficacy of different doses of buparvaquone and long- and short-acting formulations of oxytetracyclines in infection and treatment immunization using 10 -2.o dilution of Theileria parva sporozoite stabilate IL3081 Group Treatment no.
Mean days to: Schizont Fever
1
2
Buparvaquone (5.0 mgkg -~ bwt)
.
Total days of:
0
Buparvaquone (1.25 mgkg -1 bwt)
Total days of: 4
TerramycinLA ( 20 mg kg- 1bwt )
Total days of: 5
Medamycin 100 (10 mgkg-~ b w t × 2 )
Total days of: 6
No treatment
Total days of:
. 0 -
Buparvaquone (2.5 mgkg -~ bwt)
Total days of: 3
.
Recovery Death .
-
0 13.0(4)
0 18.0(3)
19.0(2)
23 16.0(2)
9 23(1)
22.5(2)
7 . 0 11.6(5) 25
-
25.0(2)
-
Positive Immune serology to in theIFAT challenge 0/5
0/5
4/5
4/5
3/3
2/3
5/5
5/5
5/5
5/5
3/3
3/3
1 .
. 0 14.4(5) 12
.
20.3(3)
20.5(2)
-, No reaction.
Figures in parentheses are the number out of five undergoing a particular reaction.
BUPARVAQUONEIN IMMUNIZATIONOF CATTLEAGAINST ~CPARVA
21
TABLE 3 The efficacy of different doses of buparvaquone infection and treatment immunization using 10- L3, 10-1.6 and 10- L8 dilution of Theileria parva sporozoite stabilate IL3081 Group Treatment no.
1
2
3
Buparvaquone ( 2.5 mg kg- 1 bwt, 10-L3 dilution of stabilate) Total days of: Buparvaquone (2.5 mg kg- 1bwt, 10-16 dilution of stabilate) Total days of: Buparvaquone ( 1.25 mg kg- 1bwt, 10- ~.8 dilution of stabilate) Total days of:
Mean days to: Schizont Fever
Recovery Death
Positive Immune serology to in thelFAT challenge
26.0(2)
-
30.5(2)
-
5/5
4/5
8 22.0(3)
0 -
25.0(3)
-
5/5
4/5
8 37( 1 )
0 -
38 ( 1 )
-
5/5
4/41
1
0
~One animal died of other causes. Figures in parentheses are the number out of five undergoing a particular reaction.
kg- 1 bwt (Table 3 ). All cattle seroconverted by Day 28 and on challenge with undiluted stabilate on Day 35, 12 out of the 14 cattle challenged were immune. DISCUSSION
The infection and treatment method of immunization depends on the use of a stabilate of wide immunogenicity containing a number of different immunogenic parasite populations infecting a large proportion of animals to be immunized (preferably 100%) while the use of chemoprophylaxis aims to prevent the development of clinical disease so that the animals become safely i m m u n e to unlimited homologous challenge (Mutugi et al., 1988b, 1989 ). It is possible to overcome the effect of the oxytetracycline treatment as it is not schizonticidal but depends on slowing down the development of the theilerial infection so that the i m m u n e response of the cow can control the infection (Spooner, 1990). Usually a balance can be obtained between the doses of sporozoite and oxytetracycline but when large numbers of animals are used, there are exceptions. This makes infection and treatment difficult to monitor in the field. Both parvaquone and buparvaquone (Clexon and Butalex, respectively Pitman-Moore) can be used in infection and treatment immunization. Par-
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P.N. N G U M I ET AL.
vaquone, if used on Days 0 or 4 after infection at its therapeutic level (20 mg kg- bwt), can block the development of parasites so that cattle do not become immune. Therefore, it has to be used on Day 8 after infection or later (Dolan et al., 1984, 1988; Young et al., 1990). On the other hand, buparvaquone treatment given on Day 0 after infection at its reported therapeutic level (2.5 mg kg-1 bwt) has been reported to allow the development of immunity (McHardy and Wekesa, 1985; Mutugi et al., 1988b, 1991 ). Hence it was believed that buparvaquone treatment would be safer than oxytetracycline treatment (Mutugi et al., 1988a) as it had schizonticidal effects when given on Day 0 but would allow the development of immunity (McHardy and Wekesa, 1985; Mutugi et al., 1988a). Therefore it was hoped that buparvaquone would prevent clinical reactions which can develop when T. parva derived from buffalo is used in immunization with oxytetracycline treatment. Unfortunately, in these experiments, where the drug was titrated against two levels of sporozoite stabilate dose of T. parva, the results were not as anticipated. When the higher dose of T. parva stabilate was used ( 10 ~ dilution), low doses of buparvaquone (2.5 and 1.25 mg kg -1 bwt) did not prevent the clinical disease as compared with the control cattle and a high proportion died. Although 2.5 mg kg-~ ofbuparvaquone did reduce the theilerial reaction, two animals still died. Buparvaquone treatment at 5 mg kg considerably reduced the clinical reaction but one animal died of theileriosis and one did not become immune. Hence there would appear to be no advantages ofbuparvaquone over oxytetracycline when used with this stabilate dose. When the stabilate dose was reduced ten-fold in Experiment 2, reactions in all groups were reduced and only two of five animals in the control group died. Hence the infecting dose appeared to be at the level of LDs0 which we have already shown with oxytetracycline treatment to be a usually effective and safe immunogenic dose of stabilate (Mutugi et al., 1988b, 1989). This was confirmed in this experiment where none of the oxytetracycline-treated animals showed theilerial reaction during immunization and all were immune to challenge with a 50-fold higher dose of homologous stabilate. The buparvaquone-treated animals showed a different pattern in the 5 mg kg-l group where none reacted, no serological titer developed and all were susceptible on challenge. It appears that this dose of drug blocked the development of infection and thus immunity with this dose of stabilate. Again in the 2.5 mg kg- 1 group, clinical reactions were prevented but one out of five animals was not immune. The 1.25 mg kg-1 dose of buparvaquone did not prevent the development of clinical reactions and one out of three surviving cattle was not immune, It was of interest that even when the stabilate dose was reduced, 1.25 mg kg- ~ bwt of buparvaquone was not effective in preventing development of clinical theileriosis. When intermediate doses of sporozoites were investigated in Experiment 3, the clinical reaction of cattle receiving 2.5 mg kg- 1 bwt of buparvaquone
BUPARVAQUONE IN IMMUNIZATION OF CATTLE AGAINST T 1~4R1~4
23
was reduced and immunity to challenge appeared to be improved, particularly with the 10-~.6 dilution of stabilate. No control cattle were used in Experiment 3 as extensive data were available from previous experiments using dilutions of 10°, 10 -1 and 10 -2 (Mutugi et al., 1988a,b). It can be concluded that using this stabilate it might be more desirable to use oxytetracyclines than buparvaquone as the therapeutic level to assure the infection of parasites with the development of homologous immunity without clinical reaction using buparvaquone treatment might be too narrow. This principle might not apply to other T. parva stabilates as indicated by McHardy and Wekesa ( 1985 ) and Mutugi et al. (1988a). The reasons for the choice of buffalo stock of T. parva for these experiments were that it provided protection against Narok District stocks of T. parva derived from buffalo in a geographically distant area (Mutugi et al., 1988b ) and because a previous stabilate prepared from this buffalo had worked well in the infection and treatment immunization at 10 -2.o dilution using either buparvaquone or oxytetracycline (Mutugi et al., 1988a,b). In the present experiments, however, neither 10 -l-° nor 10 -2.o dilutions of stabilate IL3081 worked well with buparvaquone treatment. Intermediate dilutions of stabilate appeared to work better and 10-16 dilution may be suitable for field immunization. To broaden these observations, more extensive experiments need to be undertaken. Meanwhile, treatment with either long- or short-acting formulations of oxytetracycline, for infection and treatment immunization against buffalo-derived T. parva infection with 10 -2° dilution of ST. IL3081 is recommended. ACKNOWLEDGMENTS We would like to thank Pitman-Moore, Europe for the supply of buparvaquone and their advice in experiments particularly, Dr. N. McHardy and Dr. D. Morgan. They also provided some financial support. This paper was published by kind permission of the Director of the National Veterinary Research Centre, Muguga. During these studies, Dr. A.S. Young and D. Lampard were employed by the UK Overseas Development Administration.
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P.N. N G U M I ET AL.
East Coast fever (Theileria parva infection of cattle). Evaluation of a cell culture antigen. Res. Vet. Sci., 13: 451-455. Dolan, T.T., Linyonyi, A., Mbogo, S.K. and Young, A.S., 1984. A comparison of long-acting oxytetracycline and parvaquone for immunization against East Coast fever by infection and treatment. Res. Vet. Sci., 37:175-178. Dolan, T.T., Linyonyi, A., McHardy, N., Bond, A.L. and Clampitt, R.D., 1988. Chemotherapy of East Coast fever: parvaquone treatment of Theileria parva parva at intervals after infection. Res. Vet. Sci., 44:15-20. McHardy, N., 1989. Buparvaquone, the new antitheilerial: a review of its efficacy and safety. In: T.T. Dolan (Editor), Theileriosis in Eastern, Central and Southern Africa. International Laboratory Research for Animal Disease, Nairobi, pp. 158-165. McHardy, N. and Wekesa, L.S., 1985. Buparvaquone (BW720C) - - a new anti-theilerial naphthoquinone: its role in therapy and prophylaxis. In: A.D. lrvin (Editor), Immunization against Theileriosis in Africa. International Laboratory for Research on Animal Diseases, Nairobi, p.88. McHardy, N., Wekesa, L.S., Hudson, A.T. and Randal, A.W., 1985. Anti-theilerial activity of BW720C (buparvaquone): a comparison with parvaquone. Res. Vet. Sci., 39: 29-33. Mutugi, J.J., Young, A.S., Maritim, A.C., Linyonyi, A., Mbogo, S.K. and Leitch, B.L. 1988a. Immunization using varying infective doses of Theileria parva lawrencei sporozoites derived from an African buffalo (S.vncerus caller) and treatment with buparvaquone. Parasitology, 96: 391-401. Mutugi, J.J., Young, A.S., Maritim, A.C., Ndungu, S.G., Stagg, D.A., Grootenhuis, J.G. and Leitch, B.L., 1988b. Immunization of cattle against theileriosis using varying doses of Theileria parva parva sporozoites and oxytetracycline treatments. Int. J. Parasitol., 18:453-461. Mutugi, J.J., Young, A.S., Maritim, A.C., Ndungu, S.G., Mining, S.K., Linyonyi, A., Ngumi, P.N., Leitch, B.L., Morzaria, S.P. and Dolan, T.T., 1989. Immunization of cattle against theileriosis in Coast Province of Kenya: Laboratory evaluation of a large Theileria parva parva stabilate for use in "Infection and treatment" immunization in the field. Res. Vet. Sci., 47: 170-179. Mutugi J.J., Young, A.S., Kariuki, D.P., Morzaria, S.P. and Ole Tameno, J.M., 1991. Epidemiological observations on theileriosis following field immunization using infection and treatment with either oxytetracycline or buparvaquone. Trop. Anita. Health Prod., 23: 7581. Neitz, W.O., 1955. Corridor Disease, a fatal form of bovine theileriosis encountered in Zululand. Bull. Epizoot. Dis. Afr., 3: 121-123. Spooner, P.R., 1990. The effects of oxytetracycline on Theileria parva in vitro. Parasitology, 100:11-17.
Uilenberg, G., 1976. Tick-borne diseases and their vectors. 2. Epizootiology of tick-borne disease. World Anita. Rev., 17: 8-15. Young, A.S., Leitch, B.L., Dolan, T.T., Mbogo, S.K., Ndungu, S.G., Grootenhuis, J.G. and de Castro, J.J., 1990. Evaluation of infection and treatment methods in immunization of improved cattle against theileriosis in an endemic area of Kenya. Vet. Parasitol., 35: 239-257.
