VETERINARSKI ARHIV 69 (5), 271-277, 1999

ISSN 1331-8055 Published in Croatia

Immunisation of calves against East Coast fever
without drug treatment

Onon Zanaa and Olivier Sparagano*

Centre for Tropical Veterinary Medicine, University of Edinburgh, Edinburgh, Scotland, United Kingdom

* Contact address:
Dr Olivier Sparagano,
Department of Biological Sciences, Heriot-Watt University, Riccarton Park, Edinburgh EH14 4AS, Scotland, UK,
Fax: 44 131 451 3009, E-mail: o.a.e.sparagano@hw.ac.uk

Zanaa, O., O. Sparagano: Immunisation of calves against East Coast fever without drug treatment. Vet. arhiv 69, 271-277, 1999.

ABSTRACT

A mild Theileria parva stock (T. parva Lanet, Kenya) was used for vaccination of calves prior to a lethal challenge with a heterologous stock (T. parva Marikebuni, Kenya). Although vaccination did not produce an important parasitaemia in the calves, high levels of antibodies were observed using an immuno-fluorescent assay test. All the vaccinated animals survived the lethal challenge, but none of the control animals. Protection was better when using either an autologous vaccine or a tick stabilate rather than a heterologous vaccine.

Key words: theileriosis, vaccine, calf, mild strain, Theileria parva

Introduction

East Coast fever is caused by Theileria parva, a tick-borne haemoprotozoan parasite of cattle of East and Central Africa. Ticks and tick-borne diseases, particularly East Coast fever, constitute major constraints to the dairy industries (KARIUKI et al., 1994) of the countries of East Africa, most particularly Kenya, which has 3.2 million dairy cattle (Bos taurus) of predominantly European genotype. East Coast fever kills 1.1 million cattle each year in Africa, with direct economic losses estimated at US$ 168 million (MUKHEBI et al., 1992). An infection and treatment method is mainly used and 5 to 10 % of the vaccinated animals react and need further drug treatment. This method is based on the use of live parasites, followed by a treatment with oxytetracycline to reduce the parasite proliferation and allowing the cattle to develop an immune response. A new mild T. parva stock has been recently described (MBOGO et al., 1996). Vaccinated animals are not always protected against heterologous challenge. In order to reduce animal reaction and suffering after vaccination, milk destruction after drug treatment and to check a protective effect against a lethal challenge, we tested a vaccine stock using a mild strain of T. parva.

Materials and methods

Three four-month-old male Friesian calves were used for the trial. Calves 1, 71 and 76A were vaccinated with an autologous tissue culture, a heterologous tissue culture and a tick stabilate, respectively. After 35 days all calves, including two control calves (58B and 76B) were challenged with a lethal dose of Theileria parva Marikebuni (stabilate 72 at 2 tick-equivalent/ml).

Animals were infected with the schizont form (for calves 1 and 71) and with the sporozoite form (for calf 76A) of T. parva Lanet 131 and challenged with T. parva Marikebuni tick stabilate 72, both isolated from Kenya.

The vaccine was prepared either from T. parva Lanet infected ticks or from T. parva Lanet infected tissue culture. For calf 76A, live ticks were washed in 1% benzalkonium chloride (Roccal-Winthrop Laboratories, Surrey, England) and then successively washed three times with 70% ethanol and warmed Eagles Minimum Essential Medium with Hanks salts and antibiotics (penicillin: 200 international units/ml; streptomycin: 200 µg/ml; nystatin: 100 µg/ml). Ticks were then ground up in 2-5 ml of cold 3.5% MEM/BPA (Bovine plasma albumen, Sigma - Armour Fraction V), centrifuged (100 g for 5 minutes) and the supernatant was re-suspended to have a 2.5 tick-equivalent/ml stock solution (or 13 sporoblast-equivalent) in Minimum Essential Medium/Bovine plasma albumin medium with 7.5 % glycerol (derived from BROWN, 1981). For calves 1 and 71, a tissue culture was prepared by isolating uninfected peripheral blood mononuclear cells (PBM) from calf 1 and the tissue culture infection was carried out with GUTS (grounding up tick supernatant, see above). After one week peripheral blood mononuclear cells from calf 1 were infected with T. parva Lanet 131. Cells were cryopreserved in dimethyl sulphoxide (BDH Ltd) and, when needed, were resuscitated, harvested in 1640 RPMI medium (Gibco), centrifuged, washed in fresh RPMI medium and then used for autologous (calf 001) or heterologous (calf 71) vaccination. 108 cells in 10 ml medium were injected subcutaneously (5×2 ml) for calves 71 and 1.

After 35 days post-vaccination the calves were challenged subcutaneously with 0.8 ml of 2.5 tick-equivalent of stabilate 72 above the right prescapular lymph node. Parasitaemia was read from 5% Giemsa stained blood smears from peripheral blood collected in EDTA vacutainers, and expressed as a percentage of infected blood cells. White blood cells and red blood cells were counted by using a Coulter Counter ZM and using Isoton II as the diluent using a Coulter Diluter DDIII, both apparatuses are from Coulter Electronic Ltd (Luton, UK). Zapoglobin is mixed with the diluted blood to lyse the red blood cells and count directly white blood cells. Each sample is counted twice. The packed cell volume index was calculated by using haematocrit tubes and a Hawksley MHC centrifuge. The indirect fluorescent antibody test (IFAT) was performed on antigen slides that were previously prepared with tissue culture infected with either schizonts or piroplasms of Theileria parva (Muguga). Sera were diluted from 1:40 to 1: 10,240.

Results

Parasitaemia and haematological results are presented in Table 1. The two control animals showed the lowest white blood cells and the highest temperatures, parasitaemia, and number of pyretic days. However, the results from the calf receiving a heterologous vaccine are very close to those observed for the two control animals. To avoid animal suffering, calves 76B and 58B were terminated pre-extremis at day 15 and 14 respectively. Calves 1 and 76A, receiving an autologous tissue culture vaccine and a tick stabilate vaccine respectively, showed the best protection against the lethal challenge (Table 1).

Table 1. In vivo results after a lethal Theileria parva Marikebuni
challenge in calves

WBC=white blood cells (× 103/µl); RBC=red blood cells (× 106/ µl); PCV=packed cell volume

Indirect fluorescent antibody test assay test results showed that no antibodies or low level of antibodies were present prior to vaccination. Although all three vaccinated animals showed a significant increase of antibodies with both antigens, the highest levels were observed with the piroplasm antigen as opposed to the schizont one (Fig. 1).

Fig. 1. Indirect immuno-fluorescence assay test (IFAT) results 28 days after immunisation of calves with T. parva Lanet stock

Discussion

This paper reports a successful vaccination against a lethal T. parva challenge with a recently described T. parva stock (MBOGO et al., 1996). An "infection and treatment" method is currently used for cattle vaccination against East Coast fever. Our approach could improve dramatically the cost of the vaccination (no drug to pay for and no milk to destroy after treatment) as well as providing better welfare for the animal and, therefore, building up the farmer's confidence. Other authors have tried successfully to vaccinate cattle with recombinant antigens against eimeriosis or babesiosis (BARRIGA, 1994). As far as the authors are aware, this method has not been successful and "infection and treatment" methods are still used worldwide.

Indirect fluorescent antibody test assay titres are higher with the piroplasm form, which is not surprising considering that during the Theileria parva life cycle, the schizont form is hiding in the lymphocyte and is therefore less exposed than the piroplasm form.

It will be of importance to analyse the immunological variation between these three different vaccines, and what immunoglobulin have been produced (WAGNER et al. 1974). An autologous vaccine is unrealistic in the field, but a tick stabilate is currently used in vaccinating areas. The results observed with the calf receiving an autologous cell line vaccine are disappointing, although they are similar to those found by other colleagues working on T. annulata (NICHANI et al., 1997). Over protection during a vaccination trial leads to a non-proliferation stage for the parasite, and therefore cattle do not develop any immunity. Tick stabilates are mainly used for East Coast fever but tissue culture is used for vaccination against T. annulata, while infected blood is used for babesiosis, cowdriosis and anaplasmosis (PIPANO, 1995). Although different approaches have been used against East Coast fever, it seems that tick stabilates provide better protection than any other parasite forms. However, a standardisation of the tick material in terms of parasite concentration should be established. It is important to note that in our method we did not use any chemotherapy that could prevent infection establishment, and counter-protect vaccinated animals by reducing their immune response (PIPANO, 1995). Negative effects of oxytetracyclines during a challenge have been observed when working with T. parva Boleni (HOVE et al., 1995). However, the buparvaquone effect is time-dependent (WILKIE et al., 1998 and 1998a).

We hope to continue certain investigations in order to corroborate that class I Major Histocompatibility Complex-restricted cytotoxic T lymphocyte are produced by Theileria parva Lanet vaccinated animals, as has been observed previously for cattle vaccinated with other Theileria parva stocks (MORRISON et al., 1995). However, antigenic diversity between stocks showed variation in cross protection (MORRISON 1996). The aim of future planned experiments is to check that potential cytotoxic T lymphocyte responses with T. parva Lanet provide protection against a wide range of T. parva stocks. It is also a fundamental requirement, before using this new stock as a potential vaccine, that we check its mildness throughout tick and cattle passages in the field. Other authors observed virulence variation when a mild stock is passaged through cattle (BROCKLESBY and BAILEY, 1968).

Acknowledgements
This work has been funded by a grant from the Department for International Development (formerly Overseas Development Agency), and the European Union. Ms Zanaa received a fellowship from the British Council.

References

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Received: 15 October 1998
Accepted: 3 November 1999

Zanaa, O., O. Sparagano: Imunizacija teladi protiv East Coast fever bez davanja lijekova. Vet. arhiv 69, 271-277, 1999.

SAZETAK

Za cijepljenje je rabljen blagi soj protozoa Theileria parva (T. parva Lanet, Kenya) prije izlaganja heterolognom letalnom soju (T. parva Marikebuni, Kenya). Iako cijepljenje nije izazvalo znacajnu parazitarnu invaziju u teladi, utvrdene su visoke razine protutijela putem imuno-fluorescentonog testa. Sve cijepljenje zivotinje su prezivjele invaziju letalnog soja, a nije prezivjela niti jedna od kontrolnih zivotinja. Zaštita je bila bolja kod primjene autologne vakcine ili suspenzije krpelja nego kod primjene heterologne vakcine.

Kljucne rijeci: tajlerioza, cijepivo, tele, blagi soj, Theileria parva