Population attributable fractions of farm vector tick (Rhipicephalus appendiculatus) presence on Theileria parva infection seroprevalence under endemic instability.

The primary objective of this study was to assess the impact of Rhipicephalus appendiculatus tick presence (exposure variable) on Theileria parva infection seroprevalence (outcome variable) in a group of cattle belonging to a farm using population attributable fractions (PAF). The analyses were based on a representative sample of 80 traditional smallholder mixed farms. The farms were selected by first stratifying the population administratively and implementing a multistage random sampling in Mbeere district in Kenya. The PAFs were estimated using the stratified, Bruzzi, and sequential partitioned PAF approaches. A secondary objective was, thus, to evaluate the impact of the approaches on the PAF estimates. The stratified and Bruzzi approaches estimated proportion of T. parva infection cases directly attributable to the exposure after controlling for confounding by agro-ecological zone (AEZ). The sequential partitioned PAF approach estimated a PAF associated with exposure after adjusting for any effect that the AEZ may have had by influencing the prevalence of the exposure. All analyses were carried out at the farm level where a farm was classified as infested if the tick was found on cattle on a farm, and infected if at least one animal on a farm was positive for T. parva antibodies. Variance estimation for PAFs was implemented using 'delete-a-group' jackknife re-sampling method. The stratified PAF (26.7% [95% CI: 9.0%, 44.4%]) and Bruzzi PAF (26.4% [95% CI: 9.6%, 43.2%]) were consistent in estimating a relatively low impact of farm vector tick presence with a relatively high level of uncertainty. The partitioned PAF (15.5% [95% CI: 1.5%, 29.6%]) suggested that part of the impacts estimated using the stratified PAF and Bruzzi approaches was driven by AEZ effects. Overall, the results suggested that under endemic instability in Mbeere district, (1) presence of R. appendiculatus was not a good indicator of T. parva infection occurrence on a farm; (2) ecological variation could play a role in determining infection impacts. This study provides a preliminary basis for evaluating the potential value and utility of estimating PAFs for variables amenable to control in tick-borne diseases (TBDs) epidemiological studies.

Estimating seroprevalence and variation to four tick-borne infections and determination of associated risk factors in cattle under traditional mixed farming system in Mbeere District, Kenya.

A cross-sectional study of serum antibody responses of cattle to tick-borne disease (TBD) parasites (Theileria parva, Theileria mutans, Anaplasma marginale and Babesia bigemina) was conducted on traditional smallholder mixed farms in Mbeere District in Kenya. The objective was to estimate the infections' seroprevalence and variation and identify associated risk factors. A total of 440 cattle in 80 farms, selected by stratified random sampling from the four divisions in the district, were surveyed. Information on animal and on each farm's management practices, particularly on tick control practices, was obtained by personal interview using a standardized questionnaire. Prevalences of serum antibodies were determined using the enzyme-linked immunosorbent assay (ELISA) technique. The relationship between TBDs seroprevalence and the risk factors was assessed by multivariable analysis using standard logistic regression models and mixed models using the farm as a random effect. Overall estimation of seroprevalences and their 95% confidence limits were: T. parva (19% [14%, 25%]), T. mutans (25% [20%, 29%]), A. marginale (58% [52%, 64%]) and B. bigemina (19% [15%, 23%]). Analysis in presence of extra-binomial variation under Analysis Of Variance (ANOVA) yielded relatively larger intra-farm correlation coefficient (ICC) (0.3) and variance-inflation factor (VIF) (2.35) values for T. parva than for the other parasites [range, 0.05-0.07 (for ICC) and 1.02-1.32 (for VIF)]. Both farm- and area-level variables had variably significant and large effects on all infections, but these were more pronounced on T. parva seroprevalence. Inclusion of farm random effect resulted in substantially higher estimate of farm variance component for T. parva infection (1.73) compared to other infections [range, 0.29-0.56], comparable ICC values with those under ANOVA analysis [range, 0.08-0.35] and a substantially better fit than the standard multivariable logistic regressions. The above results serve as possible indicators of existence of endemic instability for the studied TBD infections in the district. A probable differential ecological and climatic variability in vector suitability habitats, particularly for T. parva vector, was likely in Mbeere District and this was suggested to influence farm tick control management across the area. Implications of the design-based sampling and analyses on the above results are also discussed.

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.

Isolation of the causative agent of heartwater (Cowdria ruminantium) from three Amblyomma species in eight Districts of Kenya.

Thirteen isolates of Cowdria ruminantium were made from eight different Districts of Kenya by four different isolation methods. Feeding adult Amblyomma species ticks derived from nymphs collected in the field and the inoculation of homogenates prepared from adult field ticks had the highest success rate. The reattachment of adult ticks collected in the field was successful on only one of five attempts, and the subinoculation of blood from suspected heartwater carriers was unsuccessful. Seven of the isolates were derived from A variegatum ticks, four from A gemma, one from A lepidum and one from a mixed pool of the last two species. This is the first report of the isolation of C ruminantium from A gemma ticks, and the first report of its transtadial transmission from nymphal to adult A gemma.

A mild Theileria parva parasite with potential for immunisation against East Coast fever.

Twenty-three Friesian cattle were inoculated subcutaneously anterior to the left prescapular lymph node with 1 ml of a mild isolate of Theileria parva. The cattle developed low macroschizont parasitosis but no clinical reaction was observed. Thirty-five days later the cattle were grouped into five groups and challenged with five different Theileria parva isolates (four cattle-derived Theileria and one buffalo-derived Theileria). The cattle were all solidly immune to challenge with the cattle-derived Theileria isolates but three out of five of the cattle challenged with the buffalo-derived parasite died of theileriosis. All ten non-immunised control cattle developed severe theileriosis and were treated with buparvaquone (Butalex; Pitman-Moore).

Assessing the economic impact of immunisation against East Coast fever: a case study in coast province, Kenya.

The cost of immunising cattle against East Coast fever by the infection and treatment method has been calculated for a pilot scheme in Kaloleni Division of the Coast Province of Kenya by using a spreadsheet model. The cost was calculated to be KSh 544 (US$25) per animal (in 1990 values). If a farmer were to bear all this cost, immunisation would be financially profitable in grade cattle, but the benefits of immunisation would not be sufficient to justify the immunisation of zebu cattle. For these animals, the cost of immunisation would have to be in the range of KSh 230 to KSh 415 per animal, or the farm-gate price of milk would have to increase by at least 80 per cent from KSh 7.50 to 13.50/litre, or the government would have to subsidise the cost either partially or fully. The first two possibilities are realistic, because the costs of routine immunisation are likely to be lower than for the pilot scheme, and because the increasing demand for milk is likely to push up prices in the liberalised markets. If both the grade and zebu cattle in Kaloleni Division were targets for immunisation, it is estimated that there would be 14,500 head for immunisation annually, costing an estimated KSh 8 million. The spreadsheet model used to assess the economics of immunisation in the Kaloleni Division could be applied to determine the government or private veterinary service charges for immunisation that would be financially profitable to farmers in a defined cattle production system in any division, district or country. The model could also be used to estimate the annual total number of cattle for immunisation in a target cattle production system and thus help with the financial planning for the exercise.

Isolation and preliminary characterisation of a previously unidentified Theileria parasite of cattle in Kenya.

A Theileria parasite was isolated from cattle on a ranch in Kenya where it caused mild theileriosis in approximately one third of the cattle exposed to natural tick challenge. The parasite was isolated by inoculation of blood into two experimental cattle. Blood from one of these cattle was used to infect two splenectomised cattle which developed high piroplasm parasitaemias and severe anaemia. A blood stabilate was prepared from one of the splenectomised cattle and produced high parasitaemias in splenectomised cattle. Immunofluorescence tests showed that the unidentified Theileria species was distinct from other African Theileria and Babesia species of cattle. The Theileria species was shown to be antigenically distinct by means of species-specific monoclonal antibodies. The piroplasma stage was relatively large for a Theileria parasite and erythrocyte infections were usually associated with veils and bars. The salivary glands of Rhipicephalus appendiculatus, R pulchellus, R evertsi and Amblyomma variegatum fed on parasitaemic cattle did not become infected and these ticks failed to transmit the parasite. It was concluded that this parasite represented a new species of cattle Theileria in Kenya.

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.

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.

Transmission of Theileria parva by a population of Rhipicephalus appendiculatus under simulated natural conditions.

A 2 hectare paddock on the Muguga Estate, Kiambu District of Kenya (altitude 2100 m) free of Theileria parva-infected ticks was seeded by applying Rhipicephalus appendiculatus nymphs to cattle infected with Theileria parva (Kiambu 4). It was estimated that over 50 000 engorged nymphs fell onto the pasture from 4 cattle with high parasitaemias during the cold season (June). Samples of these ticks were placed in plastic cylinders under the vegetation in the paddock and sexual stages of T. parva were detected in gut lumen smears in these samples up to 21 days after repletion. Zygotes were first observed to transform into kinetes on day 55 and parasites were first detected in salivary glands of adults on day 64. Moulting of the nymphs started on day 60 after repletion and was completed by day 87. Cattle introduced into the paddock showed their first infestation with adult R. appendiculatus on day 64 after repletion of the nymphs and the infestation level gradually increased. On day 76 after repletion, 17% of a sample of adult ticks infesting cattle showed salivary gland infections with T. parva and this increased to over 70% by day 150. From day 360 onwards, a decrease in both the percentage of ticks infected and the number of salivary gland acini infected/tick was noted. In addition, T. parva infections within the salivary glands required a longer period of feeding in the older ticks before they developed into sporozoites. Cycling of Theileria through ticks was prevented by the removal of female ticks before they completed repletion. Lethal challenge levels of T. parva for cattle existed in the paddock up to day 547 after repletion, after which introduced cattle showed no infection or only a sub-lethal infection. Cattle introduced into the paddock on day 808 after repletion showed no evidence of T. parva infection. The tick infestation of introduced cattle decreased markedly from day 368 and infestation had virtually ceased by day 808.