Linkage disequilibrium between alleles at highly polymorphic mini- and micro-satellite loci of Theileria parva isolated from cattle in three regions of Kenya.

Theileria parva schizont-infected lymphocyte culture isolates from western, central and coastal Kenya were analysed for size polymorphism at 30 T. parva-specific variable number tandem repeat (VNTR) loci using a panel of mini- and micro-satellite markers. The mean number of alleles ranged from 3 to 11 at individual loci and 183 distinct alleles were observed in total, indicating high genetic diversity within the T. parva gene pool in Kenyan cattle. The frequency distribution of the length variation of specific alleles among isolates ranged from normal to markedly discontinuous. Genetic relationships between isolates were analysed using standard indices of genetic distance. Genetic distances and dendrograms derived from these using neighbour-joining algorithms did not indicate significant clustering on a geographical basis. Analysis of molecular variance demonstrated that the genetic variation between individual isolates was 72%, but only 2.3% when isolates from different regions were pooled. Both these observations suggest minimal genetic sub-structuring relative to geographical origin. Linkage disequilibrium was observed between pairs of loci within populations, as in certain Ugandan T. parva populations. A novel observation was that disequilibrium was also detected between alleles at three individual pairs of VNTR loci when isolates from the three regional meta-populations were pooled for analysis.

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes.

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. 'Deep 454 pyrosequencing' of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva.

Production in ascites fluid of biosynthetically labelled monoclonal antibody to Theileria parva sporozoites.

A hybridoma cell line has been previously produced which secretes monoclonal antibodies able to neutralize sporozoites of Theileria parva, the causative agent of East Coast fever of cattle. Cells from this line were injected intra-peritoneally into pristane-treated BALB/c mice. During the last 4 days of hybridoma cell growth, mice were given 4 daily intraperitoneal injections of a mixture of tritiated amino acids in order to biosynthetically radiolabel the monoclonal antibody being produced in ascites fluid. The specific activity of the antibody obtained was 100 mCi/mmol. The labelled antibody was used to detect, by autoradiography, a surface coat antigen of T. parva sporozoites in cryostat sections of Theileria-infected tick salivary glands. The method allows the preparation of large quantities of biosynthetically radiolabelled immunological probes for the detection of immunoreactive sites in biological specimens.

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis.

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4-7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9-10 X 10(6) base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7-8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G + C content than T. parva or the presence of highly reiterated G + C-rich regions.

A panel of microsatellite and minisatellite markers for the characterisation of field isolates of Theileria parva.

Mini- and microsatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis of parasites. Herein we describe the identification of a panel of 11 polymorphic microsatellites and 49 polymorphic minisatellites of the protozoan haemoparasite Theileria parva. The PCR products were run on high resolution Spreadex gels on which the alleles were identified and sized. The sequences of the mini- and microsatellites were distributed across the four chromosomes with 16 on chromosome 1, 12 on chromosome 2, 14 on chromosome 3 and 18 on chromosome 4. The primers from the 60 sequences were tested against all the Theileria species that co-infect cattle in East and Southern Africa and were found to be specific for T. parva. In order to demonstrate the utility of these markers, we characterised eight tissue culture isolates of T. parva isolated from cattle in widely separated regions of Eastern and Southern Africa (one from Zambia, one from Uganda, two from Zimbabwe, four from Kenya) and one Kenyan tissue culture isolate from Cape buffalo (Syncerus caffer). The numbers of alleles per locus range from three to eight indicating a high level of diversity between these geographically distinct isolates. We also analysed five isolates from cattle on a single farm at Kakuzi in the central highlands of Kenya and identified a range of one to four alleles per locus. Four of the Kakuzi isolates represented distinct multilocus genotypes while two exhibited identical multilocus genotypes. This indicates a high level of diversity in a single population of T. parva. Cluster analysis of multilocus genotypes from the 14 isolates (using a neighbour joining algorithm) revealed that genetic similarity between isolates was not obviously related to their geographical origin.

Immunization against East Coast fever: the use of selected stocks of Theileria parva for immunization of cattle exposed to field challenge.

Two antigenically different stocks of Theileria parva parva (Kilifi and Marikebuni), previously characterized as belonging to groups A and C respectively on monoclonal antibody (MAb) profiles, were selected for immunization of different breeds of cattle against East Coast fever (ECF) by the infection and treatment method. A total of 52 immunized cattle and 33 susceptible controls of different group sizes were exposed to field challenge by ticks for periods of 42-90 days at three field sites where ECF is endemic on the Kenyan coast. All immunized cattle survived ECF challenge, but 87% of the controls died of the disease. The cattle exposed at one site had been immunized 1 year earlier and maintained tick-free in the intervening period. The level of immunity in these cattle was similar to that of cattle which had been immunized 1 or 2 months prior to exposure. Thus, immunity had not waned over the 1-year period. A study at another site showed that acaricidal treatment of immunized cattle could be safely extended from twice a week to once every three weeks, whereas in susceptible cattle even twice weekly spraying did not control ECF. The isolates made from infected controls during the trials indicated the presence of three T. p. parva stocks as defined by MAb profiles. Of the two stocks used for immunization, T. p. parva Marikebuni induced broader protection. In view of the apparent limited antigenic diversity of T. p. parva strains within the Coast Province it is suggested that the Marikebuni stock might represent a key stock for vaccination in this area.

Isolation and characterization of bovine Theileria parasites in Zimbabwe.

Theilerial parasites of cattle were isolated by a variety of methods from the Harare area of Zimbabwe. Parasite stocks were established in lymphoid cell cultures and as cryopreserved sporozoite stabilates in the laboratory. Fourteen stocks in culture were characterized by testing them with monoclonal antibodies (MAb) raised against T. parva parva and T. parva lawrencei antigen. Two of these stocks had profiles similar to T. taurotragi isolates from East Africa, the other stocks had profiles similar to T. parva parva, however, many of them failed to bind MAb No. 7, and this may be a distinctive feature for T. parva bovis. Three T. p. bovis stocks were titrated by injecting different doses of the respective stabilates into pairs of cattle. Reactions ranged from severe to inapparent according to the stocks and dose used, but no fatal reactions were recorded, even at the highest dose rate. On recovery, all cattle were given homologous and then heterologous challenge. The results of the latter challenge showed that the Boleni stock gave good cross-protection against challenge with two other Zimbabwean stocks. This stock may therefore be a candidate for immunizing cattle, under field conditions, to protect them against T. p. bovis in Zimbabwe. Non-pathogenic strains of T. p. bovis may be difficult to distinguish from T. taurotragi unless cross-challenge experiments can be conducted and/or MAb profiles have been made. An improved serological test is needed to differentiate antibodies to these parasites in the sera of recovered cattle.

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.

Characterisation of stocks of Theileria parva by monoclonal antibody profiles.

Sixteen monoclonal antibodies, raised against macroschizonts of Theileria parva, were tested against 10 different stocks of the parasite. The indirect fluorescent antibody test was used to demonstrate that these antibodies showed different binding affinities to macroschizonts of the various stocks. A profile of antibody binding could thus be prepared for each stock. For a given stock the profile was consistently the same irrespective of culture passage level, host cell background and method of antigen preparation. Monoclonal antibody profiles thus appear to provide a means of characterisation of stocks of T parva in vitro, and preliminary evidence suggests that profiles may be used to differentiate strains. The best source of antigen for testing theilerial stocks was macroschizont infected cells raised in culture, but suitable preparations could also be made from lymph node biopsies of cattle infected with East Coast fever. In a field outbreak of disease it might thus be possible rapidly to characterise the strains of T parva involved and plan immunisation and control measures accordingly.

Immunisation against East Coast fever: correlation between monoclonal antibody profiles of Theileria parva stocks and cross immunity in vivo.

Stocks of Theileria parva, which had been characterised by monoclonal antibody profiles, were used to challenge cattle previously immunised against East Coast fever (ECF). When cattle were subjected to homologous challenge, or heterologous challenge with a stock of identical profile to that which had initiated immunity, they showed mild or inapparent reactions. However, when cattle were challenged with a stock of a different profile many underwent severe or fatal ECF reactions. Thus, there appears to be good correlation between cross resistance patterns in vivo and parasite differences detected in vitro by monoclonal antibodies. The results indicate that monoclonal antibody profiles can be used to characterise strains of T parva in vitro, and thus provide valuable data for planning field immunisation programmes. Now that monoclonal antibodies offer the potential of characterising theilerial parasites so precisely, the need arises for more disciplined use of terms describing parasite populations and collections. It is proposed that the rules of nomenclature devised for trypanosomes be adopted for Theileria species.

The outbreak of equine influenza in England: January 1976.

Equine influenza type 2 infections occurred in the Newmarket areas in January 1976. The disease did not spread to any extent and while this may have been due to recent vaccination it was found that not all vaccinated horses were fully protected. The virus involved showed some antigenic drift from the prototype strain A/equine/Miami/1/63 (Heq 2 Neq 2).

Oxytetracycline inhibition of mitochondrial protein synthesis in bovine lymphocytes infected with Theileria parva or stimulated by mitogen.

Oxytetracycline (OTC) significantly inhibited cytochrome c oxidase activity in bovine lymphocytes infected with Theileria parva and in uninfected mitogen-stimulated lymphocytes. The inhibitory effect was detected in vitro within 24 h of treatment with drug concentrations as low as 1 micrograms/ml. Following mitogen stimulation of lymphocytes, concentrations of 3 and 10 micrograms/ml OTC completely inhibited an increase in cytochrome c oxidase activity for 48-72 h. This inhibitory activity was considered to be due to a direct effect on lymphoblast mitochondrial protein synthesis. As a consequence, adenosine triphosphate activity was significantly reduced in lymphocytes stimulated either by infection with T. parva sporozoites or by mitogen and then treated with OTC. The results also indicated that parasite mitochondrial protein synthesis was inhibited by OTC. The activity of OTC reported in this study could explain the suppression of disease following 'infection and treatment' immunization against East Coast fever and the in vitro drug-inhibition of schizont development.

The effects of oxytetracycline on Theileria parva in vitro.

When bovine peripheral blood leucocytes were infected with Theileria parva sporozoites, immediate treatment with oxytetracycline (OTC) inhibited the development of sporozoites to mature schizonts. The extent of inhibition was dependent on drug concentration and duration of treatment. Concentrations of 5 micrograms/ml OTC, or higher, for 8 days completely inhibited the establishment of schizonts and their ability to transform host cells. A cytostatic effect on schizont-infected cell lines was found with three tetracyclines and was also demonstrated on uninfected lymphoblasts. The parasites were found to be sensitive to OTC during development to schizonts, but when mature and established within host cells, schizonts were not demonstrably affected. The infectivity of sporozoites and the binding of sporozoites to lymphocytes were not directly inhibited by OTC. The results may explain the action of tetracyclines when used prophylactically during immunization against East Coast fever, and also the reasons for the ineffectiveness of these drugs when used therapeutically during patent disease.

Genetic fingerprinting of Theileria parva using a telomeric DNA probe.

A cloned Theileria parva telomeric DNA sequence, designated pTpUtel, was used to characterize T. parva stocks and clones by hybridization to EcoRI-digested DNA. Eight of the nine T. parva stocks tested were discriminated by the telomeric restriction-fragment-length polymorphisms (RFLPs). Two isolates derived from buffalo 7014 by tick feeding on different occasions were also differentiated using the probe. The probe gave comparable results on purified piroplasm or schizont-infected lymphocyte DNA and did not cross-hybridize with uninfected bovine DNA. The telomeric restriction pattern of a cloned T. parva parasite remained identical after four passages through ticks and cattle. The telomeric sequence therefore represents a useful additional tool for analysis of theileriosis epidemiology.

A three-year evaluation of four commercial equine influenza vaccines in ponies maintained in isolation.

Ponies held in isolation for 40 months were vaccinated and revaccinated with four commercial equine influenza vaccines. Little or no HI antibody was detected after the first inoculation; second and subsequent annual revaccinations produced peak HI antibody titres between 7 and 14 days. Titres fell quickly between 14 and 28 days and less quickly thereafter. The decline of HI antibody appeared to be related more to the initial titre attained and to the period after vaccination than to the composition of the vaccine. The response to a first annual revaccination was superior to that produced by a second annual revaccination. Ether-treated antigens were required to identify primary and secondary responses to the equine-2-component of vaccines.

Humoral immune responses to Theileria parva in cattle as measured by two-dimensional western blotting.

Humoral immune responses to schizont antigens from six stocks of Theileria parva were compared by two-dimensional Western blotting using sera from cattle that had been infected with a T. parva stock or a clone. Isoelectric points of a polymorphic immunodominant molecule (PIM) of schizonts that induces strong antibody responses in cattle ranged from acidic to basic. Molecular masses (Mr) of the PIM of the respective T. parva stocks were as follows: T. parva Muguga, 86 kDa; Mariakani, 83 kDa; Marikebuni, 83 kDa; Uganda, 83 kDa; T. parva Boleni, 83 kDa; and T. parva 7014, 100 kDa. Among nine cattle infected with T. parva Muguga, four produced antibodies to a basic antigen having an Mr of 32 kDa. The PIM of T. parva Muguga, T. parva Boleni, and T. parva 7014 reacted strongly with serum obtained from an animal that had been infected with T. parva Muguga. Two-dimensional Western blotting using antischizont monoclonal antibodies enabled us to differentiate between stocks of T. parva.

Monoclonal antibody neutralizes the sporozoite stage of different Theileria parva stocks.

Monoclonal antibodies were raised against sporozoites of Theileria parva. One of these antibodies (MAbD1) neutralized the infectivity of sporozoites for lymphocytes in vitro and for cattle in vivo. Neutralization seemed to occur by blocking sporozoite entry into the cell. MAbD1 neutralized sporozoites of four unrelated stocks of T. parva, indicating the presence of a common antigenic determinant which may be important in initiating protective immunity.