J.J. Mutugi, A.S. Young, D. Lampard, S.G. Ndungu, P.N. Ngumi, S.K. Mining, A.C. Maritim, D.P. Kariuki, J.R. Awich and A. Kwena
Protozoology Division
National Veterinary Research Centre
Kenya Agricultural Research Institute
P.O. Box 32
Kikuyu, Kenya
Initial epidemiological and cross-immunity studies showed that a theilerial parasite stock, Theileria parva parva (Marikebuni), from Kilifi District, Kenya, conferred good protection in immunized cattle against lethal challenge with other theilerial stocks from the district (Irvin et al., 1981, 1983; Minami et al., 1983; Morzaria et al., 1987; Morzaria, this meeting). Following further laboratory characterization of a large stabilate (stabilate 3014) of this stock, involving titration in cattle and extensive cross-immunity studies (Mutugi et al., in press; Mutugi et al., this meeting), T. p. parva (Marikebuni) was selected for large-scale immunization in the Coast Province of Kenya. Before proceeding with immunization of smallholder cattle, pilot immunization trials were conducted on government farms in Kilifi District.
The four farms selected were the Animal Production Research Station (APRS), at Mariakani; the Coast Agricultural Research Station (CARS), at Mtwapa; and two farms, Kiswani Home Farm and Kiswani Top Farm, both near Malindi and belonging to the Agricultural Development Corporation (ADC) of Kenya. These trials involved "whole herd" immunization, in which all cattle, including calves 1 month old and older, were vaccinated. Normal farm procedures, such as deworming and vaccinations, were allowed to continue. The effect of ECF immunization on milk production was investigated in a selected group of lactating cows and comparisons were made between the immunization reactions of beef and dairy cattle and between cattle breeds.
The T. p. parva (Marikebuni) stabilate was stored in 0.5-ml aliquots in pre-labelled, colour-coded plastic straws in a portable liquid nitrogen container. Before inoculation, the plastic straws were removed from the container, rapidly thawed by rubbing them between the palms of the hands and the contents dispensed into a universal bottle. Appropriate dilutions were made using Eagle's Minimum Essential Medium, with 3.5% bovine plasma albumin and 7.5% glycerol.
Three sporozoite stabilate concentrations were used: 1:5 dilution at APRS, Mariakani; 1:10 dilution at CARS, Mtwapa and ADC Home and Top farms; and a 1:50 dilution on milking cows at Home Farm, Kiswani. A long-acting oxytetracycline treatment (Terramycin LA, Pfizer, UK) was given at 20 mg/kg (+ 10%) at the same time as stabilate inoculation at APRS, Mariakani, and CARS, Mtwapa, farms to control the immunization reaction. At ADC Home and Top farms, a comparison in the immunization treatment was made between two oxytetracycline formulations, Medamycin 100 (Tech America Group) a short-acting tetracycline, and Liquamycin LA (Terramycin LA, Pfizer, USA), a long-acting oxytetracycline. Medamycin 100 was given at 10 mg/kg on days 0 and 4 of the immunization, and Liquamycin LA at 20 mg/kg at the same time as stabilate inoculation. All drugs were injected deep into the gluteal muscles.
IMMUNIZATION TRIALS
Animal Production Research Station, Mariakani. The Animal Production Research Station farm is on the Nairobi-Mombasa Road 30 km from Mombasa. The typical animal is a Sahiwal/Aryshire cross kept on a low nutritional plane on pasture with only occasional feed supplementation given to milking cows. Disease challenge includes Trypanosoma vivax and Trypanosoma congolense infections, helminthiasis, anaplasmosis and East Coast fever. Tick control was by weekly dipping in quintiophos (Bacdip, Bayer) and trypanosomiasis was controlled by chemoprophylaxis using isometamidium chloride (Samorin, May & Baker).
Two hundred and fifty cattle over 1 month old received 1.0 ml of a 1:5 dilution of the stabilate subcutaneously below the left parotid lymph node and another 20 cattle were inoculated subcutaneously on the shoulder. All were treated simultaneously with Terramycin LA. From day 14 following immunization, clinical parameters were monitored for all cattle. From any animal with a rectal temperature of 39.5 °C or above, the parotid and prescapular lymph nodes were sampled and blood smears were taken, stained with Giemsa's stain and examined for theilerial parasites. A reacting animal also had its packed cell volume estimated and its buffy coat examined for trypanosomes, using the haematocrit contrifugation technique (Woo's Test). An animal with clinical signs of theileriosis, East Coast fever (ECF), shown by a prolonged high schizont parasitosis accompanied by fever lasting longer than 3 days, was designated an ECF reactor and treated with parvaquone (Clexon, Wellcome) and a supportive antibiotic. On day 35 after the immunization, cattle were bled and examined for T. parva antibodies using the immunofluorscent antibody test (Burridge & Kimber, 1972).
The results of serological testing are shown in Tables 1 and 2. Of 271 cattle, 260 (96%) developed T. parva antibodies following immunization. Two of these (0.7%) had ECF reactions that required treatment with parvaquone. There were seven deaths due to an haemorrhagic Trypanosoma vivax outbreak. During this immunization there was also an outbreak of foot-and-mouth disease. Serological results showed that immunized animals that suffered from severe foot and mouth lesions or clinical trypanosomiasis developed antibody titres to T. parva antigen that did not differ from antibody titres in immunized animals that did not develop these diseases. There were no obvious differences between the immunized and non-immunized cattle in their milk production.
Table 1. East Coast fever reactors following infection-and-treatment immunization on four farms at the Kenya coast
|
Farm |
Total no. immunized |
ECF reactors |
%ECF reactor |
|
Mariakani |
271 |
2 |
0.7 |
|
Mtwapa |
102 |
15 |
14.7 |
|
Kiswani Top Farm |
617 |
6 |
0.9 |
|
Kiswani Home Farm |
391 |
16 |
4.1 |
Table 2. Serological responses to Theileria parva antigen following immunization
|
|
Total no. examined |
Positive IFAT* |
Positive IFAT (%) |
|
Pre-Immunization |
|||
|
Mariakani |
254 |
28 |
11.0 |
|
Mtwapa |
89 |
18 |
20.2 |
|
Kiswani Home Farm |
139 |
26 |
18.2 |
|
Kiswani Top Farm |
197 |
46 |
23.4 |
|
After Immunization |
|||
|
Mariakani |
271 |
260 |
96.0 |
|
Mtwapa |
102 |
100 |
98.0 |
|
Kiswani Home Farm |
296 |
278 |
93.9 |
|
Kiswani Top Farm |
197 |
194 |
98.5 |
|
Kiswani milk herd |
89 |
86 |
96.6 |
* Titre greater than 1:40.
Coast Agricultural Research Station (CARS), Mtwapa. The Coast Agricultural Research Station farm is in the coconut belt 20 km north of Mombasa on the Mombasa-Malindi Road. The cattle are a pedigree Jersey breed comprising at the time of the experiment 39 lactating cows, 13 dry cows, 27 bulling heifers, 27 weaner heifers and 17 calves on milk. Disease challenge included trypanosomiasis, helminthiasis and anaplasmosis among others. A pre-immunization serological sample indicated that 20% of the cattle had T. parva antibodies. Tick control was by weekly dipping in quintiophos and isomethamidium chloride was administerd every 3 months for trypanosomiasis.
One hundred and two cattle, including calves 1 month old and older, were immunized using 1.0 ml of 1:10 dilution of T. parva (Marikebuni) stabilate given subcutaneously below the left parotid lymph node and were simultaneously treated with Terramycin LA. A selected group of milking cows (10 immunized, 10 non-immunized controls) was put on an experiment to investigate the effect of the immunization on milk production. Following immunization, the animals were monitored as described above. Serological examination showed that 100 out of the 102 cattle (98%) had developed antibodies to T. parva antigen following the immunization. However, 15 cattle (14.7%) had clinical ECF reactions, 14 of which were treated with parvaquone and recovered uneventfully. Ten of the reactors were weaners. All 17 immunized calves developed antibodies to T. parva without undergoing a clinical theilerial reaction. The results are shown on Tables 1 and 2. Immunization appeared to have no significant effects on milk production.
Agricultural Development Corporation Farms, Malindi. The Agricultural Development Corporation (ADC) has two farms in this complex: Kiswani ADC Home Farm, which is just outside Malindi, and Kiswani ADC Top Farm, some 15 km away. The Home Farm is dairy, the Top Farm predominantly beef. The farms together have a total of over 1000 cattle. Because the cattle numbers were large, the immunization operation was conducted in phases starting with the Top Farm, which becomes almost inaccessible after the rains. Both farms practice isomethamidium chloride chemoprophylaxis and regular deworming. Recently, vaccinations against brucellosis, vibriosis and leptospirosis have been instituted. East Coast fever challenge is high on both farms, especially on the Top Farm. A pre-immunization serological survey indicated that 18.7% of cattle on the Home Farm had T. parva antibodies. Just before the immunization there was an ECF outbreak on the Top Farm involving 30 cattle; 5 cattle died within a week.
Cattle were inoculated with 1.0 ml of 1:10 dilution of the stabilate subcutaneously on the shoulder (except for some milking animals) on both farms and were treated with either Medamycin 100 or Liquamycin LA. An extra 10% of drug over the calculated dose was given because most weight measurements were by weighband or visual estimation. Animals were then monitored as described above.
ADC Top Farm, Kiswani. Six hundred and fourteen beef cattle, predominantly Boran, ranging from 1-month-old calves to 15-year-old cows were immunized in two phases. In the first phase, 197 cattle consisting of young calves and dry cows were immunized. Clinical monitoring was done from days 14 to 28, and 3 out of the 197 cattle (0.5%) were identified as ECF reactors. Serological examination on day 35 showed that 190 of the 197 cattle (96.5%) had developed significant titres against T. parva antigen in the immunofluorescent antibody test. In the second immunization operation, 420 cattle consisting of heifers, dry or in-calf cows, including 16 beef and dairy bulls, were immunized on the Home Farm. Four animals (0.95%) required parvaquone treatment. One of these, a bull, acquired a natural infection, which was diagnosed two days after immunization.
ADC Home Farm, Kiswani. The Home Farm is a dairy operation of some 450 cattle of Sahiwal, Boran, Aryshire and Brown Swiss crosses. At the time of immunization, 120 cattle were lactating. The immunization was conducted in two phases. Initially, 306 cattle consisting of 92 yearlings, 32 weaners, 40 calves over 1 month old, 4 young bulls and 138 dry cows and steers were immunized using a 1:10 dilution of stabilate and treatment with either Liquamycin or Medamycin. Following immunization, 14 (4.5%) became theilerial reactors and were treated with parvaquone. Four animals died during the immunization but only 2 (0.6%) deaths were considered to be due to ECF. Serological examination on day 35 after immunization showed that 278 of 296 (95.6%) had developed T. parva antibodies.
In the second phase, 73 lactating cows and 12 young calves were immunized using 1:50 dilution of stabilate with either Medamycin or Liquamycin LA treatment. Following immunization, 2 animals, 1 calf and 1 cow (2.3%), required parvaquone treatment. Preliminary results showed no apparent effect on milk production in immunized animals. The trial continues. Serological examination on day 35 showed that 96% of the cattle had developed significant T. parva antibodies.
CONCLUSIONS
After these four trials the following observations were made on the method of immunization.
a) With the selected doses of T. p. parva (Marikebuni) stabilate, over 95% of the cattle developed antibodies to T. parva after undergoing inapparent theilerial reactions.b) Both formulations of oxytetracycline, a short-acting formulation given in two doses and a long-acting formulation given at the same time as stabilate inoculation, controlled the immunization reaction equally effectively.
c) Exotic breeds, exemplified by the Jersey cattle, had a higher number of clinical ECF reactions, suggesting an inherently lower resistance to clinical theileriosis compared to the Zebu-European breed crosses. Slightly lower stabilate doses may be recommended for immunization in exotic cattle and a second dose of short- or long-acting formulation of tetracycline may be necessary.
d) Slightly higher numbers of ECF reactors were recorded in dairy animals and weaners than in beef cattle, suggesting that the stress associated with the physiological/developmental stage of the animal may have caused a more severe immunization reaction.
e) East Coast fever immunization appeared to have no effect on milk production, except for those few animals that developed ECF during the immunization.
f) Concurrent extraneous infections with foot-and-mouth disease or trypanosomiasis did not seem to affect the development of T. parva antibodies in immunized cattle.
g) Calves 1 month old tolerated the immunization well. This augurs well for calfhood immunization, with the ideal animal being 1-4 months old.
h) In any ECF immunization trial there will be a small percentage of reactors, even with optimal stabilate concentration and drug dosage, due to individual cattle susceptiblity to T. parva sporozoites and the disease.
i) The application of infection and treatment could be speeded up by the use of shoulder inoculation of stabilate and automation of oxytetracycline injection.
ACKNOWLEDGEMENTS
We thank the directors and staff of the Coast Agricultural Research Station, Mtwapa, and the Animal Research Station, Mariakani, for facilitating these immunization trials, particularly Messrs Mureithi and Ogola, officers-in-charge of the livestock sections in Mtwapa and Mariakani, respectively. We also acknowledge the support of the management of the Agricultural Development Corporation, the Managing Director, Mr. W. Kilele, and Drs. Korir and Mutai, in charge of the Livestock Division, for permission to work on the ADC farms. We thank with special gratitude the ADC Complex farm management, the Divisional Manager, Mr. J. Purvis, and the Complex Managers Mr. W. Abila, for their co-operation throughout the trials. The trials were carried out with the permission of the Director of Veterinary Services, Kenya.
REFERENCES
Burridge, M.J. and Kimber, C.D. (1972). The indirect immunofluorescent antibody test for experimental East Coast fever (Theileria parva infection of cattle): evaluation of a cell culture schizont antigen. Research in Veterinary Science 13: 451-455.
Irvin, A.D., Chumo, R.S.C., Dobbelaere, D.A.E., Godderis, B., Katende, J., Minami, T., Ocama, J.F.R. and Spooner, P.R. (1981). Preliminary studies on East Coast fever in the Coast Province of Kenya. In: Irvin, A.D., Cunningham, M.P. and Young, A.S., eds. Advances in the Control of Theileriosis: Proceedings of an International Conference Held at the International Laboratory for Research on Animal Diseases, Nairobi, 9-13 February 1981. The Hague: Martinus Nijhoff, pp. 66-70.
Irvin, A.D., Dobbelaere, D.A.E., Mwamachi, B.M., Minami, T.P.R. and Ocama, J.G.R. (1983). Immunization against East Coast fever: correlation between monoclonal antibody profiles of Theileria parva stocks and cross-immunity in vivo. Research in Veterinary Science 35: 341-344.
Minami, T., Spooner, P.R., Irvin, A.D. and Fujinaga, T. (1983). Characterization of stocks of Theileria parva by monoclonal antibody profiles. Research in Veterinary Science 35: 334-340.
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. Veterinary Parasitology 23: 23-41.
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. (in press). Immunization of cattle against theileriosis in the East Coast Province, Kenya: laboratory evaluation of a large Theileria parva parva stabilate for use in infection and treatment immunization in the field. Research in Veterinary Science.
