Clinical efficacy and plasma concentrations of two formulations of buparvaquone in cattle infected with East Coast fever (Theileria parva infection).

East Coast fever, caused by the protozoan parasite Theileria parva, kills about 600,000 cattle annually in Africa. The hydroxynaphthoquinone compound buparvaquone (BPQ) is curative. Sixteen calves were infected with T. parva. On manifestation of disease symptoms, eight were injected with the original (pioneer) BPQ product and eight with a test product containing BPQ. All 16 calves were cured by one injection of 2.5 mg BPQ/kg bodyweight. The concentration of BPQ in blood plasma was monitored by HPLC. The mean observed C(max) of BPQ was 0.229 and 0.253 microg/mL of plasma, the mean observed time to reach this concentration (T(max)) was 2.62 and 2.12 h and the AUC (area under curve) was 4.785 and 4.156 microg h/mL, respectively, for the pioneer and test product. Considerable variations occurred in the plasma concentration of BPQ within each group. They showed no relationship with either clinical or parasitological parameters following treatment.

Some preliminary observations on the susceptibility and resistance of different cattle breeds to Theileria parva infection.

Theileria parva-naïve Friesian (Bos taurus), Boran (Bos indicus) and Maasai Zebu steers (B. indicus) were infected with a T. parva sporozoite stabilate dose which had previously been shown to induce an estimated 50% mortality rate in Boran cattle. All the cattle developed patent infections with no significant differences in the length of the prepatent period to development of macroschizonts (P > 0.05) between the three groups. Clinical theileriosis occurred in all eight the Friesians (100%), five out of nine Borans (55.6%) and two out of five Zebus (40%). Three of the Friesians (37.5%), and two of the Borans (22.2%) died of theileriosis. The different cattle types were equally susceptible to the infective dose used as indicated by the length of the prepatent periods, but there was a marked difference in their development of clinical theileriosis. The gradation in resistance to disease confirms the findings of earlier less critical studies and identifies these cattle breeds as suitable for investigations into the mechanisms of resistance to theileriosis.

In vivo comparison of susceptibility between Bos indicus and Bos taurus cattle types to Theileria parva infection.

The objective of this study was to determine whether Bos taurus cattle differ form Bos indicus in their susceptibility to infection with the Muguga stabilate of Theileria parva and in their resistance to the resultant disease. Ten Friesians (B. taurus), ten improved Borans (B. indicus), ten unimproved Borans (B. indicus) and ten Zebus (B. indicus) born to dams from an East Coast fever (ECF) endemic area were inoculated with an infective dose50 dilution of T. parva Muguga stabilate 147. All the animals except one Friesian and one Zebu developed schizont parasitosis. All the improved Borans, nine of the Friesians, eight of the unimproved Borans and six of the Zebus developed a febrile response. Four of the improved Borans, four of the Friesians and three of the unimproved Borans died of theileriosis. No significant difference (P > 0.05) in the prepatent period occurred between the groups, but the Zebus had a significantly shorter duration of schizont parasitosis (P > 0.05) and took a significantly shorter time to recover (P > 0.05) than the other three groups. There was no significant difference in the two parameters between the other three groups. The study showed that three B. indicus breds and a B. taurus breed are equally susceptible to T. parva infection. However, Zebus born to dams from an ECF endemic area showed a better ability to control the course of disease than cattle from ECF free areas.

Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization.

A sporozoite stabilate (St. 199) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.

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).

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(-1.0) dilution of stabilate was used to infect groups of cattle treated with buparvaquone at doses of between 5 and 0.625 mg kg-1 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-1 bwt or oxytetracycline gave the most satisfactory results. In the second experiment when the sporozoite dose was reduced to 10(-2.0) dilution, buparvaquone treatment at 5 and 2.5 mg kg-1 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(-1.2), 10(-1.4) and 10(-1.6) dilutions of stabilate and were treated with 2.5 mg kg-1 bwt of buparvaquone. 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-1 bwt and 10(-1.6) dilution of the stabilate would have to be carried out before such a system could be used in the field.

Immunisation of cattle against theileriosis in Nakuru District of Kenya by infection and treatment and the introduction of unconventional tick control.

One hundred and one cross European-Boran cattle (50 cows and 51 calves), on a farm in Nakuru District, Kenya, were immunised against theileriosis using Theileria parva lawrencei and Theileria parva parva stocks from another district of Kenya. The stabilates used were T.p.lawrencei (Mara III) used at 10(-1.7) dilution and T.p.parva (Kilae) used at 10(-1.0) dilution. The stabilates were combined and inoculated simultaneously with a short-acting formulation of oxytetracycline hydrochloride given intramuscularly at 10 mg kg-1 body weight and was repeated on Day 4 after inoculation of the stabilate. Most of the theileriosis challenge on the farm was thought to be derived directly from the African buffalo (Syncerus caffer). Nine percent of the cattle had significant indirect fluorescent antibody (IFA) titres before the immunisation and 99% after immunisation. The immunised cattle were exposed to tick-borne disease challenge on the farm by withdrawal of acaricide cover. The immunised cattle were divided into five groups plus two susceptible control cows and two calves for each group. Cattle in four of the groups had acaricidal ear tags, each group having a different type, applied to both ears and the fifth group remained untagged. The animals remained without conventional acaricide application for 134 days. Ten out of 20 (50%) non-immunised control cattle became T.p.lawrencei reactors which only one out of 97 (1%) of the immunised cattle reacted. A frequent complication noted was mild infections due to unidentified Theileria sp. which required expert differentiation from T.parva infections. An additional group of ten steers whose tick load was removed by hand at weekly intervals was introduced 79 days after exposure; these had no tick control and four became T.p.lawrencei reactors. Of 12 calves born during the exposure period and without tick control, four became theilerial reactors and one died. The application of acaricidal tags however, reduced tick infestation levels considerably compared with untagged controls but did not prevent transmission of theileriosis with the possible exception of tags on Group 4. A number of transient low grade fevers were noted and attributed to Theileria sp., Ehrlichia bovis, Ehrlichia (Cytoecetes) ondiri and Borrelia theileri infections, none of which were fatal. One immunised animal died of acute dual infection of Babesia bigemina and Borrelia theileri after acaricide control by spraying was re-introduced but no Anaplasma infections were detected. An analysis of the economic effects of immunisation was made.

Evaluation of infection and treatment methods in immunization of improved cattle against theileriosis in an endemic area of Kenya.

Five experiments were carried out to determine the efficacy of immunization against theileriosis in an endemic area of Kenya using artificial infection with a mixture of stabilates of Theileria parva stock or natural infection and treatment with parvaquone or several formulations of oxytetracyclines. For the first four experiments, introduced, susceptible Sahiwal/Friesian crosses were used and in the fifth, calves of Boran/Maasai zebu crosses born on the site. Cattle were infected either artificially with sporozoite stabilates of local isolates of T. parva parva derived from cattle and T. parva lawrencei derived from African buffalo or exposed to natural tick challenge on the ranch mostly derived from buffalo. The cattle were then given various treatment regimens using either parvaquone or long- and short-acting formulations of oxytetracycline. Treatment of natural infections, although it can be effective, was not considered a practical method on a large scale because of the need for intensive monitoring in the case of parvaquone treatment and the possibility of cattle not becoming infected in the case of prolonged application of long-acting formulations of oxytetracycline. Both methods were relatively expensive. Artificial infection treatment proved more practical and methods were developed where the monitoring of cattle was not required during the immunization procedure. Out of a total of 16 drug regimens investigated, one (consisting of two treatments of a short-acting formulation of oxytetracycline at 10 mg kg-1 body weight on Days 0 and 3 or 4 after infection) was found to be the most efficacious and the cheapest, and has now been used on a routine basis. This method can be used successfully on calves greater than 1 month of age.

Immunisation of cattle against theileriosis in Coast Province, Kenya: laboratory evaluation of a Theileria parva parva stabilate for use in 'infection and treatment' immunisation in the field.

Theileria parva parva Marikebuni stock, previously shown to give good protection to immunised cattle in Kilifi District, Coast Province of Kenya, was chosen for large scale immunisation in the district. A large sporozoite stabilate was prepared and evaluated for efficacy and safety in the 'infection and treatment' method, using a long or short acting formulation of oxytetracycline. Susceptible cattle were infected with selected doses of stabilate (10(0), 10(-1), 10(-1.7) and left either as untreated controls, or treated with one of the two oxytetracycline formulations. It was concluded that stabilate dilution at 10(-0.7) or 10(-1) in combination with either formulation of oxytetracycline would effect satisfactory immunisation. The short acting oxytetracycline treatment was judged to be the most efficacious in protecting cattle against homologous challenge. On heterologous challenge it was found that T p parva Marikebuni immune cattle were protected against seven T p parva stocks from Kilifi District and also against four stocks of T p parva from other areas of Kenya. In addition, the Marikebuni stock provided partial protection against challenge by T p lawrencei stocks. Furthermore, cattle immune to T p parva and T p lawrencei were protected against lethal challenge of T p parva Marikebuni stock. Thus, it appears that large scale immunisation of cattle against theileriosis in Kilifi District could be undertaken using the Marikebuni stock. With continued assessment, this stock could provide a master theilerial stock for immunisation against cattle theileriosis in areas free of buffaloes elsewhere in Kenya.

Theilerial parasites isolated from carrier cattle after immunization with Theileria parva by the infection and treatment method.

Groups of cattle were immunized with 10(-2) dilutions of sporozoite stabilates of Theileria parva lawrencei derived from African buffaloes either alone or in combination with Theileria parva parva derived from cattle and concomitant treatment with either long or short-acting formulations of oxytetracyline. At 90 or 120 days after infection, uninfected Rhipicephalus appendiculatus nymphal ticks were applied to individual immunized cattle and the resultant adults ticks were applied to individual susceptible cattle. Theilerial infection developed from ticks fed on 6 out of 11 animals investigated for evidence of a carrier state. Two additional animals were shown by cell-culture isolation to have persistent theilerial infections. Nine cattle infected with the parasites from carrier animals were treated with paravaquone and 7 recovered. These recovered cattle were then challenged with the original immunizing stabilates at 10 degrees dilution together with the original immunized and carrier cattle. Six out of 7 cattle which had recovered from carrier-derived infection succumbed to this challenge and died but none of the original immunized cattle showed theilerial reactions. When a carrier-derived sporozoite stabilate was used to challenge cattle immune to the original immunizing parasite, they proved to be immune. Cattle immune to the carrier-derived parasites were all immune to challenge with the original parasite. A monoclonal antibody profile against T. parva schizonts isolated by cell culture from samples of the experimental animals did not appear to be sensitive enough to determine the antigenic differences between the carrier-derived parasite and the original immunizing parasite. Indications are that the carrier state is not likely to produce new antigenic strains which would be dangerous to immunized cattle.