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Thirty-eight sera from cattle from a herd in Rhodesia naturally infected with Theileria lawrencei were titrated with the indirect immunofluorescent test against schizont antigens of T parva (Muguga) and T lawrencei from Rhodesia. T lawrencei was found to be indistinguishable from T parva with the test.
Precipitating antigens from Theileria parva have been partially purified. Two antigens from each of the schizont and piroplasm stages of the parasite were identified; the major antigens from the two stages shared the same specificity. The antigens showed considerable molecular heterogeneity, almost certainly a result of the preparative method, and they always contained large amounts of DNA. The piroplasm antigens were of parasite nuclear origin and the schizont antigens were probably of the same origin. The antigens were weakly antigenic, and the activity against them of humoral antibody from cattle immune to East Coast fever was low. These antigens do not appear to induce protection against East Coast fever.
Cell-free schizonts and merozoites of Theileria parva and Theileria lawrencei, derived from parasitized lymphoid cell lines, were propagated in vitro using enriched medium. Use of radioisotopic markers showed that the bovine cell-independent parasites passed through a limited but marked replication by day 4. If normal bovine RBC were inoculated in vitro with the cell-free merozoites, development of piroplasms in the RBC occurred. Electron microscopic studies of the merozoites and piroplasms revealed their structure to be similar to previous descriptions. Cattle inoculated with T parva merozoites and schizonts and later challenge exposed with homologous stabilate developed leukopenia, showed low macroschizont index, and survived longer than unvaccinated controls.
The developmental stages of Theileria parva within the intestine of ticks were studied by electron microscopy. On the 2nd-4th day post repletionem (p.r.) the intestine of the ticks contained numerous stages, which were very similar to microgamonts and microgametes of haemosporidia. From these results it seemed likely that gamogony of Theileria parva takes place in the intestine of ticks, too. The microgamont-like stages were spear-head-like; they measured about 10.5 mum in length with a maximum diameter of about 2.1 mum in their middle region. These parasites, which were limited by a unit membrane, had a stiletto-like apex, several flagellar-like protrusions of about 3 mum in length and mostly two slender posterior protrusions. The stiletto-like structure was electron-dense and measured about 2.0 mum in length; at its base it appeared spongy consisting of coiled, fibrillar elements of about 20 nm in diameter. Up to 4 flagellar-like protrusions were found in cross sections originating near the base of the stiletto-like structure. At most, up to six microtubules were seen within these protrusions, which, however, at their free ends usually contained only two microtubules. Near the base of the stiletto-like structure two groups of additional microtubules began. One group ran beneath a third of the peripheral margin to the posterior cell pole. The other group was bundle-like arranged. In cross sections through the posterior slender protrusions of the parasite about 10 microtubules appeared in an irregular pattern. The slender posterior protrusions had nearly double dimensions of the flagellar-like protrusions.
Methods for infecting Rhipicephalus appendiculatus ticks with Theileria parva by injection and by artificial feeding were confirmed and compared. The injection method proved simpler and at best as effective and suggested improvements are described.
Cattle were inoculated with dilutions of Theileria parva (Muguga) stabilate without concurrent antibiotic treatment. The dilutions were selected to induce severe East Coast fever infections, which in some cases would lead to death. Serum samples were collected and the complement fixation (CF), indirect haemagglutination (IHA) and indirect immunofluorescence (IFA) tests were used to detect T. parva serum antibodies. Sera were fractionated on Sephadex G200 to determine the activities of specific immunoglobulin fractions. Sequential production of IgM antibody followed by 7SIg antibody was demonstrated by the IHA and CF tests. All IFA activity was restricted to the 7SIg fraction. Serologic reactions in the IHA test were detected as early as 4 days after inoculation of the stabilate, CF activity after 8 days and IFA activity after 21 days.
Rhipicephalus evertsi successfully transmitted fatal East Coast fever (Theileria parva infection) in one experiment. A second one gave negative results, as did an attempted transmission of T. PARVA BY Amblyomma variegatum.
Initial laboratory trials of an experimental subunit vaccine against Theileria parva based on the 67-kDa major sporozoite surface antigen revealed a range of responses to challenge. We have analyzed convergence in seven sets of monozygotic twins which suggests that genetic factors may have an influence in determining the degree of protection provided by p67 immunization. In addition, we have examined whether allelic diversity at major histocompatibility complex class II loci influences protection. Analysis of bovine leukocyte antigen DRB3 diversity in 201 animals identified significant associations with vaccine success (DRB3*2703; P = 0.027) and vaccine failure (DRB3*1501; P = 0.013). Furthermore, DRB3*2703 was associated with the likelihood of immunized animals showing little to no clinical signs of disease following challenge. We discuss the acquired and innate immune mechanisms that may be behind the associations described here.
Bovine lymphoblastoid cell cultures permanently infected with Theileria parva and T. annulata were used in an in vitro screen to test a wide range of compounds for chemotherapeutic activity against these parasites. Only two compounds, menoctone and methotrexate, showed significant activity, which has subsequently been confirmed by tests in infected cattle. Menoctone was about 100 times more active than methotrexate. Mixtures of menoctone and various other other compounds were tested, but showed no potentiation. The activity of menoctone was antagonized by cycloguanil and methotrexate. The in vitro test represents a rapid, cheap, and apparently reliable method of screening compounds for activity against Theileria.
Theileria parva piroplasm infection of mouse erythrocytes was detected, following intraperitoneal inoculation of mice with irradiated cultures of T. parva-infected bovine lymphoid cells. Infection could not be transmitted to cattle using Rhipicephalus appendiculatus ticks.
It was demonstrated that Theileria parva parasites infective to cattle could be induced by exposure of infected adult Rhipicephalus appendiculatus ticks to 37 degrees C. The production of the sporozoites of T parva was not as efficient as by feeding ticks on rabbits.
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.
In a series of experiments, groups of cattle, undergoing patent East Coast fever (ECF) reactions induced by the inoculation of tick-derived stabilates of Theileria parva infective particles, were treated by parenteral administration of 2 tetracyclines. Ten cattle were treated with n-pyrrolidinomethyl tetracycline at 15 mg/kg for 5 days, starting on the first day macroschizonts were detected in lymph node biopsy smears. Nine of these treated cattle survived ECF reactions induced by an inoculum which killed 7 of 10 untreated controls. The same tetracycline, at 10 mg/kg daily for 5 days from the first day of febrile response had no effect on the disease. Oxytetracycline was administered at 15 mg/kg daily for 5 days to 2 groups of cattle infected with ECF. All 5 cattle survived which were treated on the first day parasites were seen. Four of 5 survived in which treatment was delayed till the onset of febrile response. All 5 untreated controls in this experiment died.
A study was made of the effects of serum and gamma-globulins from vaccinated cattle (donor cattle) given to cattle (principals) exposed to Theileria parva infection (East Coast fever (ECF)). The principals were treated with normal serum, with serum containing high antibody titer against T parva (as evaluated by the indirect fluorescent antibody (IFA) technique), or with globulins prepared from normal and from immune serums. For exposure, the principals were inoculated with standardized suspension of the tick Rhipicephalus appendiculatus containing T parva infective particles. Comparison was made between treatments with normal serum and globulin and treatments with immune serum and globulin, using observations of time lapse after exposure to febrile response (39.5 C), to appearance of T parva macroschizonts, and to death. In addition, hematologic changes were determined. Neither the establishment of infection nor the clinical and hematologic changes in the principals were influenced by their treatment with immune serum or concentrated globulins; all died of ECF. Humoral antibodies did not protect cattle against fatal ECF, nor was enhancement of T parva infection observed.
Cattle were immunized against East Coast fever by the inoculation of a tick-derived stabilate of Theileria parva infective particles and the intramuscular injection, at daily intervals, of n-pyrrolidinomethyl tetracycline at a dose level of 5 mg/kg. Four doses of the tetracycline, starting on the day of inoculation of the infective stabilate, minimised the ECF reactions in the cattle during immunization. Immunity to homologous T. parva stabilate challenge was demonstrated in 27 out of 27 cattle infected and treated in this manner.
The development of motile stages, called kinetes, from a stationary stage (regarded as zygote) has been followed in Theileria parva by means of electron miscroscopy. This process started after moult of the tick nymphs which had sucked on highly infected calves, i.e. about 20 days after repletion (a.r.) of the ticks. The transformation took place by formation of a growing protrusion (= anlage) into an inner, enlarging vacuole. During this process the limiting membrane of the enlarging vacuole serves as the outer membrane of the developing motile stage, whereas the two inner ones as well as the subpellicular microtubules are newly formed. This transformation proceeds rapidly, so that on the 25th day a.r. most of the kinetes have already left the gut cells and started penetration into the salivary gland cells. On the way to the salivary glands nuclear divisions occurred within the kinetes. The steps of the transformation described were compared to those in T. annulata and to ookinete formation in haemosporidia.
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