Characterization of species and strains of Theileria.

A variety of methods is now available for characterizing species and strains of Theileria. For many practical purposes involving field control of theileriosis, characterization on a broad basis may be sufficient, but in other areas much more precise characterization is required. Such precision can be usefully exploited only when cloned parasite populations are involved, and methods to improve parasite characterization and parasite cloning should be developed concurrently. The current methods of immunization against theileriosis involve the use of live parasite populations which are generally poorly defined and, in addition, have the capacity to undergo biological change (by selection, mutation or genetic recombination) within hosts and vectors. Such changes may be difficult to define and identify, but could have profound effects on immunization strategies. Improved methods of parasite characterization and selection, which are now becoming available, will enable parasite stocks for immunization to be identified and selected more precisely, and any biological changes that occur can be monitored. Improved methods of parasite characterization will also open the way to a better understanding of Theileria genetics and the mechanisms of heritability, which appear to differ in some fundamental ways from patterns of Mendelian inheritance. Controlled matings between selected and defined populations of parasites can be envisaged, with the aim of producing hybrid parasites for immunization. In addition, the prospects of modifying the theilerial genome by genetic manipulation become very real: transfection vectors tailored by restriction enzymes could be used to insert or modify gene sequences to develop parasites with appropriate sets of characters. It may also be possible to identify parasite genes which trigger the cytotoxic response which is so important in immunity (Eugui and Emery, 1981; Emery et al., 1981; Preston et al., 1983). Such genes might then be transfected into bovine host lymphocytes to generate immunity against the whole parasite (Iams, 1985). The gene products which are responsible for stimulating immune responses could also be synthesized artificially and used for vaccination. Methods of characterizing Theileria range from Giemsa's staining to DNA hybridization; all have a role to play, and by judicious selection of appropriate methods for particular circumstances, it is becoming possible to characterize theilerial parasites very precisely. Improved methods of characterization can, in turn, lead to a better understanding of parasite biology and to the development of improved methods of immunization and control.

Effect of timing and intensity of challenge following immunization against East Coast fever.

When groups of Theileria parva parva Muguga-immunized cattle were given a homologous lethal challenge at different times after immunization, it was found that 4/6, 5/6, 6/6 and 6/6 animals survived when challenged on Days 5, 10, 20 and 30, respectively, post-immunization. With a heterologous challenge (T.p.parva Marikebuni), 2/6, 5/6, 4/6, 4/6 and 5/6 cattle survived when challenged on Days 5, 10, 20 and 30, respectively, after immunization. All controls, except one, died of East Coast fever (ECF). The survivor underwent severe ECF and recovered after a prolonged convalescence. When two T.p.parva Muguga-immunized animals were each given homologous challenge by application of 1000 infected ticks (infection rate of 20 infected acini (i.a.) per tick), both survived a mild ECF reaction. When groups of T.p.parva Muguga- or T.p.parva Muguga/Marikebuni-immunized cattle were challenged with different doses of T.p.parva Muguga sporozoites (equivalent of 140, 1400 and 14,000 i.a. per animal), 28/29 cattle survived. All controls died of ECF. It was concluded that cattle could be safely exposed to tick challenge 1 week after immunization by infection and treatment using appropriate immunizing stock(s). Massive homologous challenge did not break through the immunity induced by the immunization procedure.

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.

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.

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.

Age resistance to Theileria parva bovis infection in calves.

Data collected in the Zimbabwean province of Mashonaland-West, in the period 1980-1988, showed that mortality in calves owing to Theileria parva bovis infection (January disease) was significantly lower in animals younger than 7 months than in older cattle. Groups of seven Holstein-Friesian calves from non-immune dams aged approximately 1, 4, 7, 10 and 13 months were infected with a Theileria parva bovis tick-derived stabilate. The dose chosen was lethal for 40% of the calves in the trial. Mortality was highest in the 4-month age group. The reactions in the 7-, 10- and 13-month age groups became progressively milder. The reactions in the 1-month old calves were the least marked, being very mild. The age-related resistance in the youngest calves, as can be concluded from our results, is only of short duration and cannot explain the lower incidence of January disease observed in calves in the field.

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.

Introduction and multiplication of bovine Babesia in human cells.

Bovine erythrocytes parasitised with Babesia major were fused with human HeLa cells by means of Sendai virus. B major parasites entered the HeLa cell cytoplasm and in some cases underwent multiplication over a period of three days.

Comparison of the effect of drugs on Babesia in vitro and in vivo.

It was previously shown that Babesia-infected blood maintained in vitro incorporated high levels of 3H hypoxanthine and that the uptake could be suppressed by babesicidal drugs. In the current paper the hypoxanthine pathway was shown to be the most sensitive in testing such drugs for activity in vitro. Other pathways involving orotic acid, glutamine or thymidine were less sensitive. The highest counts were achieved when 3H hypoxanthine was added 1 h after cultures were set up. Drugs which had previously shown activity against Babesia in vitro were added to cultures of B rodhaini and after 24 h incubation parasitised cells were inoculated into groups of mice. Known babesicidal drugs suppressed or delayed infection in mice and 3H hypoxanthine analogues also showed some activity. These analogues, however, showed no therapeutic properties when injected into B rodhaini infected mice.

An ultrastructural study of heterokaryons derived from Theileria parva-infected bovine lymphoblasts and Ehrlich ascites tumour cells.

An ultrastructural study was made of Sendai virus induced heterokaryons derived from Theileria parva-infected lymphoblasts and Ehrlich ascites tumour cells. When fusion occurred, parasites were successfully integrated into the cytoplasm of the resulting heterokaryons where they appeared as morphologically normal macroschizonts. Homokaryon formation was also noted. This occurred frequently between Ehrlich ascites tumour cells and rarely with lymphoblasts. A small proportion of heterokaryons contained altered forms of T parva showing nuclear components, vesicular structures and paired organelles similar to the microshizont stage of the parasite.

The effects of irradiation on Babesia maintained in vitro.

Blood infected with Babesia radhaini, B major or B divergens was irradiated to different absorbed doses between 0 and 120 krad, and then maintained in vitro in the presence of 3H hypoxanthine for 24 h. The effects of irradiation were measured by the ability of the parasites to incorporate 3h hypoxanthine and, in the case of B rodhaini, by the ability of the parasites to infect mice. B major and B divergens were slightly more radio-resistant than B rodhaini, but all showed a progressive fall in ability to incorporate 3H hypoxanthine with increasing doses of irradiation, except at low doses of irradiation when there was increased uptake of 3H hypoxanthine. In the case of B rodhaini there was close correlation between the ability of the parasites to incorporate 3H hypoxanthine and their infectivity for mice. Both types of activity were abolished at doses of 40 krad and above.

Possible enzyme deficiency in bovine lymphosarcoma cells grown in vitro.

A culture of bovine lymphosarcoma cells was unable to grow in HAT medium, suggesting that the cells were unable to use either exogenous hypoxanthine or exogenous thymidine. Further work showed that they were able to incorporate tritiated thymidine but not tritiated guanine. This indicated that failure to grow in HAT medium was due to the cells being deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase which is responsible for incorporation of the bases hypoxanthine and guanine. This finding may have application in the therapy of bovine lymphosarcoma.

Attempts to infect T lymphocyte-deficient mice with Babesia species of cattle.

Nu/nu, nu/+, splenectomised nu/nu and Lasat mice were inoculated with freshly collected bovine blood infected with Babesia divergens and B major. There was no evidence that either parasite became established in mice but B divergens persisted in mice up to 10 days whereas B major lasted only one day. B divergens infection generally persisted longer in splenectomised mice but absence of thymus made no apparent difference to persistence of infection. B divergens underwent morphological changes in mice to vacuolated and ring forms.

Attempts to produce Theileria parva-infected mouse cells using cell fusion techniques.

Ehrlich ascites tumour (EAT) cells were fused with Theileria parva-infected bovine lymphoid (C2) cells using inactivated Sendai virus. A variety of techniques were employed to try and increase fusion percentage and harvest of heterokaryons. There was no evidence that pre-exposure of C2 cells to Sendai virus or phytohaemagglutinin improved fusion percentage, but the percentage was higher when 10(8) C2 cells were fused with 10(7) EAT cells, as compared with a ratio of 10(7) C2: 10(6) EAT. An increased yield of multinucleate cells was obtained using a calf serum gradient to separate cells of different sizes. An attempt to fuse EAT cells with free macroschizonts was not sucessful. When cells were grown in hypoxanthine/aminopterin/thymidine medium (HAT), T parva appeared to be selectively killed by the aminopterin present; this finding would seem to militate against the use of a HAT selection system to clone parasitised hybrids. C2 cells could be selected out by passage of mixed cultures through mice, but no evidence of parasitosis was detected in EAT cells which became established.

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.

Serological comparison of British Theileria mutans and Japanese T sergenti.

A serological comparison between British Theileria mutans and Japanese T sergenti using the indirect fluorescent antibody test, showed that the two parasites were indistinguishable. On the basis of this and previous findings it is suggested that the British parasite is identical with the Japanese one and that its name should therefore be changed to T sergenti.

Transplantation of bovine lymphosarcoma cells to athymic (nude) mice.

Bovine lymphosarcoma cells, previously established in culture, were inoculated subcutaneously into groups of irradiated and non-irradiated athymic (nude) mice. Tumours developed at the site of inoculation in all of the irradiated mice but in none of the others. Tumour growth was progressive in all cases but there was no evidence of invasion of surrounding tissues nor of metastasis. Tumour cells were passaged directly to further mice and a similar growth pattern was recorded. The use of this system suggests a possible small animal laboratory model for bovine lymphosarcoma.

Hybrid cells, infected with Theileria parva, formed by fusion of hamster and mouse cells with parasitised bovine lymphoid cells.

Bovine lymphoid cells from a culture line parasitised with Theileria parva were fused to mouse heart (MH) and baby hamster kidney (BHK) cells, grown as monolayers, using inactivated Sendai virus. The resultant heterokaryons contained macroschizonts of T parva. Macroschizonts also occurred in cells that apparently contained only BHK or MH nuclei. Parasites underwent varying degrees of development; in some cases microschizonts resulted, and in other cases massive atypical parasite masses were formed. Fusion occurred more readily between hamster and bovine cells than between mouse and bovine cells.

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.

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.