The epidemiology of Theileria parva infections on smallholder dairy farms in Kenya.

The purpose of the study was to characterize the differences in epidemiology (risks of infection, morbidity, mortality) and potential control of East Coast fever (ECF) between the selected strata. Evidence of Theileria parva infection was assessed by increased antibody levels as measured in an indirect ELISA test by the percent positivity (PP) of serum samples relative to a strong positive reference serum. A prospective cohort study was conducted in five purposively sampled agroecological zone (AEZ)-grazing system strata in Murang'a District, Kenya, between March 1995 and June 1996. The study strata were selected to represent the widest range of ECF risks in the district and included, zero-grazing and open-grazing farms in the Upper Midlands (UM) one and four AEZs and zero-grazing farms in the UM2 AEZ. In total, 225 calves from 188 smallholder farms were examined from birth to age six months. Calves were recruited into the study at birth and visited within the first two weeks of life and thereafter at biweekly intervals for up to 14 visits. Important differences were observed between the different AEZ-grazing strata. Seroconversion risks of T. parva were highest in the UM4-open grazing stratum. Antibody prevalence in adult cattle and ECF morbidity and mortality risks were also highest in this stratum. In the open-grazing strata, particularly in the lower elevation AEZ, UM4, there was stronger challenge and a greater impact of ECF. There is likely to be an expansion of smallholder dairy farming into this area so that it is likely to be the most important target production system for ECF control in the central highlands of Kenya.

Comparison between bloodstream and procyclic form trypanosomes for serological diagnosis of African human trypanosomiasis.

Trypanosoma brucei brucei MiTat 1.2 bloodstream and corresponding procyclic forms, as well as procyclics of T. b. gambiense and T. b. rhodesiense, were fixed, in suspension, using a mixture of 80% acetone and 0.25% (v/v) formalin in saline and used as antigens for diagnosis of African human trypanosomiasis by the indirect immunofluorescent antibody test. T. b. brucei bloodstream forms detected 41/42 and 37/41 parasitologically diagnosed cases of T. b. rhodesiense and T. b. gambiense trypanosomiasis, respectively, while the procyclic stages detected the same number (41/42) of T. b. rhodesiense, but fewer (29/41) T. b. gambiense infections.

Effect of chronic trypanosomiasis on immunization against East Coast fever.

Two experiments were carried out in which uninfected cattle, or cattle chronically infected with Trypanosoma congolense, were immunized by the infection and treatment method against East Coast fever (ECF; Theileria parva infection). Chronic trypanosomiasis did not prevent cattle mounting an effective immunological response to ECF immunization and resisting subsequent lethal challenge. There appeared to be no difference in the level or quality of immunity between uninfected cattle and trypanosome-infected cattle. Thus, T. congolense infection on its own does not appear to provide a constraint to ECF immunization in the field.

The prevalence of serum antibodies to tick-borne infections in cattle in smallholder dairy farms in Murang'a District, Kenya; a cross-sectional study.

The most important tick-borne disease of cattle in eastern, central and southern Africa is East Coast fever (ECF) caused by Theileria parva and transmitted by the tick Rhipicephalus appendiculatus. Other less-important tick-borne diseases in cattle are benign theileriosis caused by Theileria mutans, babesiosis caused by Babesia bigemina, anaplasmosis caused by Anaplasma marginale and cowdriosis caused by Cowdria ruminatum. In Murang's District, Central Province of Kenya, five agroecological zones (AEZs) are defined according to climate, altitude and agricultural activities. A cross-sectional serological study was conducted on 750 smallholder dairy farms in Murang's District, selected in a stratified random sampling method. The farms had a total of 362 calves. One hundred and fifty farms were studied from three administrative sublocations in each of the five AEZs. Prevalence of serum antibodies to three tick-borne parasites, that is T. parva, T. mutans and B. bigemina, were determined using the enzyme-linked immunosorbent assay (ELISA) technique. Antibody prevalence values differed across the AEZs. The ranges of means for the prevalences were: T. parva (18-72%), T. mutans (1.5-28%) and B. bigemina (12-49%). The above results serve as indicators of the possible existence of endemic stability in some AEZs for some parasites.

Evaluation of an ELISA for detection of antibodies to Babesia bovis in cattle in Australia and Zimbabwe.

An enzyme-linked immunosorbent assay (ELISA) for antibodies to Babesia bovis was evaluated in comparison with the indirect fluorescent antibody test (IFAT) in Australia and Zimbabwe. Positive and negative threshold values for the ELISA were set using sera from cattle of known infection status. Sensitivity and specificity estimates for the ELISA based on 158 positive sera from cattle experimentally infected with Australian isolates of B. bovis and 318 negative sera collected from B. bovis-free herds in Australia were 100% and 99.4%, respectively. The specificity of the assay in Africa, based on 328 sera from B. bovis-free herds in Kenya and South Africa, was 99.7%. The ELISA was compared with the IFAT using sequential sera from 16 calves experiencing primary B. bovis infections, and a total of 777 field sera collected from B. bovis-endemic herds in Australia and Zimbabwe. In primary infections, the ELISA and IFAT detected antibodies at or about the same time. With sera from endemic herds, the performance of the ELISA was at least comparable with that of the IFAT. Two hundred and fourteen of 221 sera that were negative by IFAT, were negative by ELISA, and 428 of 439 sera that were clearly positive by IFAT were positive by ELISA. Of 117 sera that gave equivocal (suspect or weak positive) results in the IFAT, 20 were positive by ELISA, 7 were suspect and 90 were negative. We conclude that the ELISA will be useful for epidemiological studies on B. bovis in Australia and Zimbabwe, and probably elsewhere.

Indirect fluorescent antibody test for experimental and epizootiological studies on East coast fever (Theileria parva infection in cattle). Evaluation of a cell culture schizont antigen fixed and stored in suspension.

A schizont antigen for the indirect fluorescent antibody test against Theileria parva was prepared from a T parva-infected bovine lymphoblastoid cell line by fixing the cells in suspension with a mixture of acetone and formaldehyde. The antigen was stored in suspension in phosphate buffered saline for one and a half years at -60 degrees C without loss of activity; the antigen could also be lyophilised. The fluorescence of the intracellular schizonts was bright and specific with T parva positive bovine control serum and absent with negative bovine control serum and Theileria mutans positive bovine control serum. Fluorescence of the lymphoblastoid cell itself was observed with Trypanosoma brucei positive control serum and some bovine test sera: this fluorescence, which masked the intracellular schizonts, was eliminated by absorbing the sera in the supernatant of sonicated lymphocytes obtained from bovine lymph nodes. The antigen was evaluated with sera from cattle experimentally infected with T parva. In an epizootiological study on East Coast fever in the Coast Province of Kenya, there was good correlation between the serological responses of cattle to T parva schizont antigen and the distribution of Rhipicephalus appendiculatus ticks.

Observations on blood-borne parasites of domestic livestock in the lower Juba region of Somalia.

The commonest parasite to be found in blood of cattle in the Lower Juba Region is Theileria mutans. Antibodies against Trypanosoma spp. and Anaplasma marginale could be detected serologically whereas no antibodies against T. parva and T. annulata were present. A list of ticks collected between 1980 and 1982 in the above mentioned area shows that vectors of T. mutans are common. As the cattle throughout the region are indigenous Somali Boran it seems that endemic stability is maintained and mortality from T. mutans infection is negligible. It is not yet clear whether pathogenic strains of T. mutans similar to those isolated in East Africa occur.

A new method for fixation and preservation of trypanosomal antigens for use in the indirect immunofluorescence antibody test for diagnosis of bovine trypanosomiasis.

A new method for fixation of trypanosomes for use in the indirect immunofluorescent antibody test (IFAT) is described. The method involves fixation of live trypanosomes, in suspension, using a mixture of 80% cold acetone and 0.25% formalin in saline. The fixed trypanosomes were stored in suspension at -60 degrees C, 4 degrees C or at room temperature for at least one year without loss of antigenicity. Using trypanosomes prepared this way as antigens in IFAT, species-specific antibodies were detected within 2 weeks of infection in sera of cattle infected with T. brucei or T. vivax. Thereafter, antibodies recognizing antigens common to the two species as well as T. congolense were detected. The antibody levels to common antigens of the three species declined 2-3 months post-infection, leaving only the species-specific antibodies. The sera obtained from T. congolense infected animals, did not react with T. vivax or T. brucei antigens. The non-specific fluorescence commonly associated with this assay was eliminated by prior absorption of the test sera with normal bovine lymphocyte lysate. This treatment of serum did not affect the specific antibodies to trypanosomal antigens. Analysis of bovine sera prepared from cattle in Kisiwani and Muhaka, along the Kenya coast, using the fixed trypanosomes revealed that some animals had antibodies to one trypanosome species only while others had antibodies to 2 or all 3 trypanosome species.

The persistence of Theileria parva infection in cattle immunized using two stocks which differ in their ability to induce a carrier state: analysis using a novel blood spot PCR assay.

An improved Theileria parva DNA detection assay based on the polymerase chain reaction (PCR) using primers derived from the 104 kDa antigen (p104) gene was developed to detect parasite DNA in blood spots on filter paper. The specificity of the assay was validated using DNA from a wide range of cattle-derived and buffalo-derived stocks of T. parva. DNA of T. annulata, T. buffeli, T. lestoquardi, T. mutans and T. taurotragi was not amplified using the p104 primers. The detection threshold of the assay was approximately 1-2 parasites/microl of infected blood. PCR amplification using the p104 primers was applied to sequential samples from groups of cattle experimentally infected with either the T. parva Marikebuni stock that induces a long-term carrier state or the Muguga stock, which does not induce a carrier state. The study extended for up to 487 days post-infection and PCR data from defined time points were compared with parasitological microscopy and serological data, together with xenodiagnosis by experimental application of ticks. Microscopy first detected piroplasms between days 13 and 16 after infection whereas all cattle became PCR +ve between days 9 and 13. Animals infected with the Muguga stock of T. parva had parasite DNA in the peripheral blood, which could be detected by PCR, for between 33 and 129 days post-infection in different animals. By contrast parasite DNA in the blood of cattle infected with the Marikebuni stock could be detected consistently from day 9 up to 487 days, when the study terminated. The data suggest that the nature and persistence of the carrier state may differ markedly between different T. parva parasite stocks.

Identification of a Theileria mutans-specific antigen for use in an antibody and antigen detection ELISA.

Purified piroplasms of Theileria mutans were used to immunize BALB/c mice to generate monoclonal antibodies (MoAbs). The MoAbs recognized an antigen of a relative molecular mass of 32 kDa in Western blots. This antigen was also recognized by sera from cattle which had recovered naturally from experimental tick-transmission or infections induced by the blood stages of T. mutans. The MoAbs did not react, in indirect immunofluorescence or enzyme-linked immunosorbent assays (ELISA), with the common haemoparasites of cattle, namely, T. parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma congolense, T. vivax or T. brucei. An antigen capture ELISA was established with two of the MoAbs which recognized different epitopes on the 32 kDa molecule. Using this test it was possible to detect circulating antigens or immune complexes in sera collected from cattle during the acute or chronic phases of infection. When the purified 32 kDa protein was used as antigen in a micro-ELISA to detect circulating antibodies in both experimental and field cattle sera, it was found that the titres of antibodies ranged between 1:20 and 1:10,240. Results of this study indicate that the antigen and immune complex capture assays and the antibody detection ELISA can be complementary in the immunodiagnosis of acute and chronic T. mutans infections. Moreover, the tests are useful in the differential diagnosis of the disease and for epidemiological studies.

Immune depression in African trypanosomiasis: the role of antigenic competition.

The capacity of trypanosome-infected cattle to mount an immune response to a simultaneous or subsequent challenge with other trypanosomes was investigated using various clones of Trypanosoma congolense and T. brucei. In animals infected simultaneously with equal numbers of trypanosomes of two different clones, the variant-specific antibody response to one clone was severely depressed while the response to the other was not affected. In cattle infected with one clone and then subsequently challenged severely depressed depending on the time interval between the two inoculations. These observations were consistent regardless of whether the clones of trypanosomes used were derived from the same or different species. The characteristics of these responses would suggest that the inability of trypanosome-infected cattle to respond well to a simultaneous or subsequent challenge with other trypanosomes or other antigens may be due to antigenic competition.

Differences in the epidemiology of theileriosis on smallholder dairy farms in contrasting agro-ecological and grazing strata of highland Kenya.

A prospective cohort study was conducted in five purposively-sampled agro-ecological zone (AEZ)-grazing system strata in Murang'a District, Kenya, between March 1995 and June 1996. The study strata were selected based on a preliminary characterization study to represent the widest range of risks to East Coast fever (ECF) in the District and included zero-grazing and open-grazing farms. In total, 225 calves from 188 smallholder farms were examined from birth to 6 months of age and visited within the first 2 weeks of life and thereafter at bi-weekly intervals for up to 14 visits. The purpose of the study was to characterize the differences in epidemiology (risks of infection, morbidity and mortality) and potential control of ECF between the selected strata. Evidence of Theileria parva infection was assessed by increased antibody levels as measured in an indirect ELISA assay by the percent positivity (PP) of serum samples relative to a strong positive reference serum. Sero-conversion risks of T. parva were highest in the open-grazing strata. Antibody prevalence in adult cattle and ECF morbidity and mortality risks were also highest in open-grazing strata. While different, all five AEZ-grazing strata were considered to be endemically unstable for ECF. East Coast fever challenge was low in all zero-grazing strata and this challenge is likely to remain low due to continuing intensification of smallholder farming in the central highlands. In the open-grazing strata, there was higher challenge and a greater impact of ECF.