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.

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 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.

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.

Monitoring Theileria parva infection in adult Rhipicephalus appendiculatus ticks.

A rapid method is described for preparing and staining salivary glands of Rhipicephalus appendiculatus ticks infected with Theileria parva. The technique, involving the use of a modified methyl green pyronin stained minimizes the risk of losing material and allows examination of stained glands within minutes of preparation. The technique was applied in a series of studies in which ticks were either infected with T. parva under different conditions, or maturation of parasites in adult ticks was stimulated by different means. When nymphal ticks were fed on the ears of cattle the subsequent infection rate of the adult ticks showed no correlation with the parasitaemia of the cattle at the time of nymphal engorgement. There was no difference in infection rates between adult ticks in which parasite maturation had been stimulated either by incubation at 37 degree C or by feeding on rabbits. However, parasite maturation took about 1 day longer in incubated ticks than in rabbit-fed ticks. Female ticks were consistently more highly infected than males, both in terms of the percentage of ticks infected and the mean number of infected acini/tick. Ticks were infected with T. parva by injection of nymphs with parasitaemic bovine blood, but the resultant adult infection was lower than that in ticks which had been infected naturally by feeding on cattle.

Glucose phosphate isomerase polymorphism in Theileria annulata and T. parva.

Six stocks of Theileria annulata isolated from the Sudan and nine stocks of T. parva, isolated in Kenya and Malawi were grown in bovine lymphoblastoid cell lines. Lysates prepared from the infected cultures were examined electrophoretically on thin layer starch gels for evidence of glucose phosphate isomerase polymorphism. The six stocks of T. annulata showed major variations in their parasite enzyme patterns but no variation was detected in nine stocks of T. parva.