Cytotoxic T lymphocytes from cattle immunized against Theileria parva exhibit pronounced cross-reactivity among different strain-specific epitopes of the Tp1 antigen

The protozoan parasite Theileria parva causes a usually fatal disease in cattle, known as East Coast fever. Cattle can be vaccinated by injecting live parasites simultaneously with long acting oxytetracycline (the infection and treatment method, ITM). The immunity induced by ITM is believed to be mediated by cytotoxic T lymphocytes (CTL). Although effective, the ITM vaccine has disadvantages such as the need for a liquid nitrogen cold chain and a complex production process, which may be overcome by the development of a subunit vaccine. However, the high level of antigenic polymorphism among different strains of T. parva may hinder the development of a subunit vaccine aimed at induction of a protective CTL response. In this study, the CTL cross-reactivity among T. parva strains was examined. The Tp1(214-224) epitope has previously been shown to be recognized by cattle of the A18 BoLA type. Three different variants of this epitope have been identified from different T. parva strains. Here, bulk CTL and CTL clones were generated from two animals using both the live sporozoite vaccine composed of three different strains and a Muguga strain for immunization. The cross-reactivity of these CTL with the three variant Tp1 epitopes was examined in interferon gamma ELISPOT assays and CTL killing assays. CD8(+) cells from both animals cross-reacted with the three variant CTL epitopes in interferon gamma ELISPOT assays, although the CD8(+) cells from the Muguga-immunized animal showed a more epitope restricted response. Clones from the vaccine immunized animal showed diverse response patterns with clones responding to each variant peptide. Although some variability in the cytotoxic response was observed, overall strong cross-reactivity among the variant Tp1 epitopes was seen in both animals. Such epitope polymorphism does not, in this case, serve as a potential challenge in a putative subunit vaccine as it would be sufficient to only include one of the variant epitopes.     

Cloned Theileria parva produces lesser infections in ticks compared to uncloned T. parva despite similar infections in cattle

Experimental transmissions of cloned Theileria parva in cattle with Rhipicephalus appendiculatus ticks were compared to transmissions with uncloned T. parva during studies on the potential for genetic recombination during syngamy of Theileria to produce antigenic diversity for evasion of bovine immunity. Prevalence and abundance of T. parva infection in adult ticks, which resulted from the feeding of nymphs on the calves, were significantly higher in the uncloned compared to the cloned T. parva. Development of sporoblasts of T. parva in the ticks to produce infective sporozoites was similar. There was no statistically significant difference in the clinical course of infection in cattle between cloned and uncloned T. parva. It was concluded that cloned T. parva has characteristics that reduce its viability during the tick stages of its life cycle.     

Evaluation of a TaqMan real-time PCR for the detection of Theileria parva in buffalo and cattle

A real-time PCR assay based on TaqMan probe chemistry was developed for the detection of Theileria parva DNA in blood samples. It uses a Theileria genus-specific PCR primer set and a T. parva-specific probe to amplify and hybridize with a species-specific part of the 18S rRNA gene of the parasite. The test was evaluated using positive and negative reference blood samples and shown to be specific for T. parva. Analytical sensitivity was determined by testing a dilution series of T. parva positive blood. It was shown to be able to detect parasitaemia as low as 2 × 10(-6)%. The Taqman assay results were also compared with that obtained with the real-time hybridization probe PCR assay, which is currently employed as the official test for the diagnosis of T. parva infections in buffalo and cattle and was shown to be equally sensitive. A panel of 1164 field samples was screened using both assays and 164 samples tested positive in both tests, indicating a good correlation.     

In vitro infection of bovine alloreactive cytotoxic T cell lines with sporozoites of Theileria annulata and T parva

Bovine alloreactive cytotoxic lymphocyte (CTL) lines of known target specificity were infected in vitro with sporozoites of Theileria annulata and T parva and cultured in limiting dilution. The phenotypes of the CTL lines both pre- and post infection were assessed using a panel of monoclonal antibodies specific for defined bovine lymphocyte subpopulations. The effector function of the resultant infected cell lines was determined using a Cr51 release assay and compared to the uninfected control CTL line. The results indicated that T parva sporozoites consistently infected and transformed the CTL lines very efficiently even at the lowest cell doses. In contrast the T annulata sporozoites were largely unable to infect and transform the alloreactive CTL except at the very highest cell and sporozoite doses. A factor which appeared to influence susceptibility to T annulata infection was an increased level of class II expression on the CTL line. None of the cell lines showed cytotoxic effector function after infection with either T annulata or T parva sporozoites.     

Chemotherapy of Theileria parva lawrencei infections in cattle with halofuginone

Halofuginone lactate, given once orally at a dosage rate of 1,2 mg/kg body mass on the 1st, 3rd or 5th days of fever, resulted in the recovery of only 1 out of 5 splenectomized cattle. Three splenectomized animals, treated on the 1st as well as the 4th day of fever, recovered and were then carriers. Six untreated controls all died. The potential value of a chemotherapeutic agent for Theileria parva lawrencei infections in South Africa is discussed.     

Geographical information systems for studying the epidemiology of cattle diseases caused by Theileria parva

Data on selected variables which influence the epidemiology of cattle diseases caused by Theileria parva were assembled and entered in a computerised geographical information system. Variables studied included the distributions of major hosts (cattle and buffalo), the vector ticks (Rhipicephalus appendiculatus and related species) and the reported presence of East Coast fever, corridor disease and January disease. In addition, the distribution of climatic suitability for R appendiculatus was assessed using the model CLIMEX run on an interpolated climate database developed for Africa. Distribution maps of each variable were produced. The potential value of geographical information systems in studies of disease epidemiology and control is discussed, with examples of how sensitivity may be enhanced by the inclusion of additional variables. In addition, subject areas in which poor data quality and inadequate data standardisation may limit the use of these systems are identified and discussed.     

Immunization of cattle with a Theileria parva bovis stock from Zimbabwe protects against challenge with virulent T.p. parva and T.p. lawrencei stocks from Kenya

Following inoculation of 34 Bos indicus (Boran) cattle with a Theileria parva bovis (Boleni) stock from Zimbabwe, 18 animals underwent mild theilerial reactions, 12 underwent moderate reactions, three suffered severe reactions and one died. When these animals were subsequently challenged with different virulent stocks of either T.p. parva (Muguga, Marikebuni or Mariakani) or T.p. lawrencei (Ngong 1 or Nanyuki) from Kenya, all except two animals resisted challenge. The two reactors were part of the group challenged with the T.p. parva (Mariakani) stock. All 12 susceptible control animals underwent severe reactions and 11 died. The results of these experiments suggest that T.p. bovis (Boleni) may be used in some situations to immunize cattle against East Coast fever without the need to provide concomitant chemotherapy as in the infection and treatment method of immunization.     

Improved immunogenicity of novel baculovirus-derived Theileria parva p67 subunit antigens

East Coast fever (ECF) in cattle is caused by the tick-borne protozoan parasite Theileria parva. The major sporozoite surface antigen of T. parva (p67) is an important candidate for inclusion in a subunit vaccine. Recently, we reported the expression and production of different parts of p67 as fusions to either GFP or to the baculovirus GP64 envelope glycoprotein in insect cells, which resulted in stable proteins recognized by a monoclonal specific for native p67. The immunogenicity of these fusion proteins was examined in out-bred mice and cattle. In mice, the full length p67 molecule without its signal peptide and transmembrane region, but fused to GFP (GFP:p67deltaSS) was the best immunogen followed by the C-terminus of p67 fused to GP64 (GP64:p67C). These two immunogens also provoked a high level of sero-conversion in cattle when formulated in a water-in-oil or saponin-derived adjuvant with only 100 microg of protein and a single booster. The vaccine-elicited antibodies efficiently inhibited the infectivity of T. parva sporozoites in in vitro neutralization assays. This study demonstrated that these new baculovirus-derived p67 vaccines were highly immunogenic, and that in combination with a suitable adjuvant, they have a clear potential to induce protective immunity in cattle.     

Evaluation of an enzyme immunoassay for serodiagnosis of infections with Theileria parva and T annulata

An enzyme linked immunosorbent assay (ELISA) was used to determine antibody levels in cattle infected with Theileria parva and T annulata, using antigens prepared from the intra-erythrocytic piroplasm stage of the parasites. Antibody levels in calves infected with T parva increased from the 16th day after infection to reach peak values at days 28 to 35 and then declined rapidly, but in calves infected with T annulata antibody levels rose steadily up to day 40. Similar patterns of antibody production were shown by indirect fluorescent antibody tests. Sera from animals infected with T parva gave higher ELISA values with the antigen prepared from the homologous parasite species than with the antigen prepared from T annulata, but sera from cattle infected with T annulata gave similar high ELISA values with antigens prepared from both T parva and T annulata. Sera from animals infected with T mutans, T sergenti, T velifera, Babesia divergens, B major and B bovis gave only slight or no cross reactions with the piroplasm antigens, but serum from a calf infected with B bigemina cross reacted at a significant level with both piroplasm antigens.     

In vitro proliferative and cytotoxic responses of PBL from Theileria annulata-immune cattle

Peripheral blood lymphocytes were isolated from healthy calves and were subsequently infected with sporozoites of Theileria annulata in vitro. The infected cells were passaged for 50 times and thereafter inoculated into animals from which they were previously isolated. Within 4-5 days, schizont-containing cells were demonstrable in the lymph nodes of all animals. Few days later, merozoites were detected in erythrocytes. A slight decrease in the counts of lymphocytes and leucocytes was also found. After 2 months these animals and a group of uninfected calves were heavily infected by tick-infestation and showed severe symptoms of theileriosis with 60% schizont-containing cells in the lymph nodes and a parasitaemia of about 35%. Because of the severity of the infection, all control calves were treated with Halofuginone. In contrast, the initially immunized cattle (by inoculation of culture cells), survived the infection without chemotherapy. Less than 10% of their lymph node cells contained schizonts, whereas less than 1% of their erythrocytes were found to be infected with merozoites. In all immunized animals, specific cytotoxic PBL, with the capacity to lyse autologous but not allogeneic infected cells, were demonstrated. In addition, a population of PBL were found to be able to inhibit the growth of T.annulata-infected culture cells in vitro. However, in comparison to PBL of immune animals, PBL of acute infected calves were superior in their capacity to inhibit the proliferation of schizont-containing cells. In mixed lymphocyte reactions, T. annulata-infected cells could induce a more pronounced proliferative response in PBL from immune than in PBL of uninfected animals.     

Immunization of cattle against Theileria parva bovis and their exposure to natural challenge

Theileria parva bovis isolates were tested for their immunizing capacity under natural field challenge on Willsbridge Farm in the highveld of Zimbabwe. Fifteen susceptible Sussex yearlings were immunized with the Boleni stock and 15 with a mixture of three isolates from the farm, using tick-derived sporozoite stabilates. No chemoprophylaxis was used. A dose of 0.1 ml of stabilate appeared to be safe in preliminary laboratory experiments, but the reactions were severe in the Sussex cattle and one died despite treatment. Twenty-nine immunized animals and 10 controls first experienced a mild infection, starting about 15 days after their arrival at the farm. Ten of the immunized animals and four controls had schizonts in peripheral lymph nodes for variable periods; one third of those had pyrexia. Nymphal Rhipicephalus appendiculatus ticks applied to three of the reacting immunized calves transmitted Theileria taurotragi to two animals and T. parva to a third. A second Theileria infection, due to T. parva bovis, was detected shortly after the first one. Schizonts were detected in seven out of 10 controls. Pyrexia was more severe and prolonged. Two of the controls died of theileriosis. At the same time schizonts were seen in three immune animals and eight of them had short periods of pyrexia. Intercurrent infections with Babesia bigemina, Borrelia theileri and Eperythrozoon were detected and may have contributed to the fever. Tick infestations were low during the exposure. In the second year of exposure, four out of eight new control animals had severe reactions, and one died. None of the immunized animals became ill, but one animal from the first year control group, which had not reacted previously, had clinical theileriosis. It is concluded that immunization provided an effective protection against field challenge.     

Laboratory and field investigations into the Theileria parva carrier-state in cattle in Zimbabwe.

The Theileria parva carrier-state in cattle on commercial farms on Zimbabwe was investigated using parasitological and serological methods. The proportion of cattle showing Theileria piroplasms on two farms, which had recent histories of disease outbreaks, were 64% (n = 106, total of heifers and weaned calves examined) and 71.5% (n = 60) while the proportion of T. parva antibodies for the same animals were 59% and 98.5%, respectively. On four farms where no cases of the disease occurred for over 10 years, the average proportion of animals showing piroplasms and antibodies were 55.4% (range 32-82, n = 223) and 73% (range 47-91, n = 223), respectively. However, on another three farms which had no history of theileriosis outbreaks these proportions were very low, being 11.4% (0-24, n = 157) for piroplasms and 12.2% (5-23, n = 157) for antibodies. The mean infection rate in unfed Rhipicephalus appendiculatus adults collected from farms with a high prevalence of cattle which were carriers of Theileria piroplasms during the tick activity season was 29% (range 12-60%) with 9.3 (range 2-18.7) mean infected acini per infected tick. The infectivity of different tick batches to susceptible cattle produced a wide spectrum of theileriosis reactions. Laboratory controlled experiments were carried out to study the persistence of T. parva (Boleni) piroplasms in cattle immunized with this strain as well as its infectivity for ticks and its subsequent transmissibility to cattle. Examination of the salivary glands of 15 batches of ticks collected from six immunized cattle on three different occasions over 18 months showed that none were infected with Theileria parasites. However, the infectivity of other ticks in the same batches to susceptible animals was demonstrated 6, 10 and 18 months after cattle had been immunized with Boleni stabilate.     

Epidemiology of tick-borne diseases of cattle in Zimbabwe. III.Theileria parva group

Summary A survey of the occurrence of antibodies toTheileria parva using the IFA test in calves up to one year old at 244 localities in Zimbabwe revealed that the parasite occurred throughout the country although the prevalence of positive serological reactors was generally low. Outbreaks of theileriosis in high rainfall areas in the north, east and west of the country were attributed toTheileria parva bovis transmitted from cattle to cattle byRhipicephalus appendiculatus. Outbreaks in high and low rainfall areas in the south and west of the country were attributed toTheileria parva lawrencei transmitted from buffalo to cattle byRhipicephalus zambeziensis orR. appendiculatus. R. appendiculatus was not uniformly distributed in Zimbabwe. It occurred very commonly in foci in the commerical farming areas but was rare in most overgrazed communal farming areas. Outbreaks of disease attributed toT. parva bovis were recorded in some but not all theR. appendiculatus foci. The disease was present in areas infested withR. zambeziensis but it did not cause cattle deaths in these areas.

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Resumen Un reconocimiento de la ocurrencia de anticuerpos deTheileria parva en terneros jóvenes hasta de un a no, llevado a cabo en 244 localidades de Zimbabwe boereveló, que el parásito se encuentra diseminado en todos pais, a pesar que le prevalencia de reactores positivos fue baja. Los brotes de Theileiriosis en las áreas lluviosas y húmedas en el norte, este y oeste del pais fueron atribuídes aTheileria parva laurencei, transmitida de búfalos a ganado bovino, por la garrapataRhipicephalus zambeziensis oR. appendiculatus. ElR. appendiculatus no se encontró uniformemente distribuído en Zimbabwe; se encontró preferentemente en explotaciones comerciales, pero no en áreas comunales de pastoréo. Brotes atribuíbles aT. parva bovis, se enconta?ron en focos deR. appendiculatus. La enfermedad, e estaba presente en áreas infestadas conR. zambeziensis, pero no causó mortalidad. Se consideran datos seroepidemiológicos, en la formulación de regímenes de vacunación para anaplasmosis y babesiosis.

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Résumé Une enquête concernant l'incidence des anticorps àTheileria parva chez des jeunes veaux de 0 à 1 an a été menée au Zimbabwe dans 244 localités. Le test de l'immunofluorescence indirecte a été utilisé. Cette enquête a montré que le parasite est présent dans tout le pays bien que la fréquence de réactions sérologiques positives soient généralement faible. Les épidémies de theileriose dans les zones à forte pluviosisté du nord, de l'est et l'ouest du pays sont attribuées àTheileria parva bovis transmits de bovin à bovin parRhipicephalus appendiculatus. Les épidémies dans les zones à forte et faible pluviosité du sud et de l'ouest du pays sont attribuées àTheileria parva lawrencei transmis du buffle aux bovins parRhipicephalus zambeziensis ouR. appendiculatus. La répartition deR. appendiculatus n'est pas uniforme au Zimbabwe. La présence est fréquente en foyers dans les zones d'élevage industriel mais elle est rare dans la plupart des zones surpaturées d'élevage traditionnel. Des épidémies de la maladie atribuée àTh. parva bovis ont été entegistrées dans quelques foyers àR. appendiculatus mais pas dans tous. La maladie était présente dans les zones infestées parR. zambesiensis mais elle n'a pas entrainé la mort des bovins.

     

Molecular characterization of live Theileria parva sporozoite vaccine stabilates reveals extensive genotypic diversity

The current Infection and Treatment Method of vaccination against East Coast fever comprises an inoculation of live Theileria parva sporozoites and simultaneous administration of oxytetracycline. Immunization with a combination of parasite types has been shown to provide broader protection than inoculation of individual strains. In this study, we used a high-throughput capillary electrophoresis system to determine the genotypic composition of the Muguga Cocktail, a widely used vaccine stabilate derived from three seed stabilates-Muguga, Serengeti-transformed and Kiambu 5. Five satellite markers were used to genotype the vaccine and reference stabilates from two commercial-scale preparations of the vaccine. In addition, 224 cloned cell lines established by infection of bovine lymphocytes with T. parva parasites from the component stabilates were genotyped. The results indicate that, for the recently prepared batch, there are at least eight genotypes in each of the Muguga and the Serengeti-transformed stabilates, while parasites from the Kiambu 5 stabilate showed no diversity at the five loci. The Serengeti-transformed stabilate contained parasites of the Kiambu 5 genotype and of two genotypes present in the Muguga stabilate, whereas there were no genotypes common to the Muguga and Kiambu 5 stabilates. When stabilates from the two vaccine batches were compared, no allelic variations were identified between the Muguga and Kiambu 5 parasites, while lack of sufficient clones prevented a full comparison of the Serengeti-transformed stabilates. The findings will facilitate examination of the extent to which the vaccine strains become resident in areas under vaccination, the identification of 'breakthrough' strains and the establishment of the quality assurance protocols to detect variations in the production of the vaccine. The cloned cell lines will be useful for further understanding the antigenic diversity of parasites in the vaccine.     

Characterisation of Theileria parva isolates from Kiambu district, Kenya

Four Theileria parva isolates from Muguga area of Kiambu district, Kenya, were used to establish schizont-infected cell lines. Their protein antigens were then separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS page). The isolates were subsequently subjected to protein analysis and characterisation by the western immunoblotting technique. Probing for the polymorphic immunodominant molecule (PIM) was done using monoclonal antibody no. 4. SDS page detected up to 20 protein antigens of molecular mass 35,000-180,000 Da. The western blot analysis revealed a greater heterogeneity in the molecular mass (M(r)) of PIM than previously thought. The M(r) of PIM varied between 80 and 90 kDa. The isolates further revealed different densities of surface epitopes with variable reaction to the monoclonal antibody. The implications of these findings to the epidemiology of east coast fever and immunisation programmes are discussed.     

Clinical and diagnostic features of East Coast fever (Theileria parva) infection of cattle

East Coast fever is a tick-borne protozoal disease affecting cattle in a large part of East and Central Africa. Since the vector occurs over an even wider range there is considerable potential for the disease to spread to countries which are currently disease free. This article, describing the clinical and diagnostic features of East Coast fever, may remind authorities in these countries of the potential hazards posed by the disease.     

Cycle of bovine lymphoblastoid cells parasitised by Theileria parva

The events were studied which occurred during different stages of the cell cycle of bovine lymphoblastoid cells infected with the parasite Theileria parva. The mean number of nuclei in macroschizonts was about 16 for cells in interphase and 30 for those in metaphase. Pulse labelling with 3H thymidine showed that macroschizonts normally incorporated thymidine when the host cell was in early mitosis. Thymidine incorporation by macroschizonts thus occurred at a different stage in the cell cycle to that when the cell nucleus incorporated thymidine in S phase. DNA synthesis by host cell nucleus and macroschizont is thus asynchronous. Division of macroschizonts appears to follow immediately after they have synthesised DNA without a G2 period. This division occurs while the host cell is in metaphase. When the cell divides each daughter cell thus contains its interphase complement of macroschizont nuclei. Some macroschizont division may occur in interphase but this is relatively insignificant when compared with that which occurs in host cell metaphase. This work suggests that T parva regulates its own DNA synthesis independently of the cell. This finding could have application in developing strategies for chemotherapeutic attack on the parasite.