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.     

Immunisation of cattle against theileriosis in Coast Province, Kenya: laboratory evaluation of a Theileria parva parva stabilate for use in 'infection and treatment' immunisation in the field

Theileria parva parva Marikebuni stock, previously shown to give good protection to immunised cattle in Kilifi District, Coast Province of Kenya, was chosen for large scale immunisation in the district. A large sporozoite stabilate was prepared and evaluated for efficacy and safety in the 'infection and treatment' method, using a long or short acting formulation of oxytetracycline. Susceptible cattle were infected with selected doses of stabilate (10(0), 10(-1), 10(-1.7) and left either as untreated controls, or treated with one of the two oxytetracycline formulations. It was concluded that stabilate dilution at 10(-0.7) or 10(-1) in combination with either formulation of oxytetracycline would effect satisfactory immunisation. The short acting oxytetracycline treatment was judged to be the most efficacious in protecting cattle against homologous challenge. On heterologous challenge it was found that T p parva Marikebuni immune cattle were protected against seven T p parva stocks from Kilifi District and also against four stocks of T p parva from other areas of Kenya. In addition, the Marikebuni stock provided partial protection against challenge by T p lawrencei stocks. Furthermore, cattle immune to T p parva and T p lawrencei were protected against lethal challenge of T p parva Marikebuni stock. Thus, it appears that large scale immunisation of cattle against theileriosis in Kilifi District could be undertaken using the Marikebuni stock. With continued assessment, this stock could provide a master theilerial stock for immunisation against cattle theileriosis in areas free of buffaloes elsewhere in Kenya.     

Identification of a target population for immunisation against East Coast fever in coastal Kenya

Two experiments were carried out to identify the target population of cattle for immunisation against East Coast fever (ECF) using the infection-and-treatment method. Firstly, a sentinel-calf study was used to determine the age window for ECF immunisation by determining ages at clinical detection of infection with Theileria parva. Six groups of five naive cross-bred (Bos taurus/Bos indicus) male calves, introduced at intervals of 2 months at a mean age of 26 days, were exposed to natural tick challenge on a high ECF-risk, small-holder farm in the coastal lowland, coconut-cassava agro-ecological zone of coastal Kenya. Secondly, a challenge study evaluated the relationship between the presence of T. parva antibodies and immunity. Ten indigenous adult Zebu cattle and nine Zebu young stock purchased from farmers in the same zone, and eight cross-bred calves (survivors of the sentinel-calf study) were challenged with 10 times the immunising dose of T. parva Marikebuni stock. Twenty-four of these 27 cattle had high antibody titres before challenge. Two cross-bred calves, obtained from an ECF-free area and seronegative to T. parva schizont antigen, also were challenged and used as susceptible controls. Twenty-five (83%) of the 30 sentinel calves contracted ECF over an age range of 36-116 days (mean 72 days). The remaining five calves died of other causes within 2 months of arrival on the farm. Fourteen of the 25 calves survived the infection and developed antibodies to T. parva. Despite tick control, seven of these 14 calves had a second episode of ECF and two died. In total, 13 of the 25 calves that contracted ECF died. Only one of 19 indigenous Zebu animals developed clinical ECF when challenged with T. parva Marikebuni (mild clinical signs with spontaneous recovery). Of the eight cross-bred survivors from the first experiment, only one succumbed to ECF when challenged and it died. Both susceptible cross-bred calves developed severe clinical signs of ECF and one died. The experimental studies show that in the high ECF-risk areas of the coconut-cassava zone of coastal Kenya, immunisation against ECF in cross-bred (B. taurus/B. indicus) cattle should be targeted at an early age (preferably within 1-2 months of birth).     

Immunization of cattle against East Coast fever using Theileria parva (Marikebuni) and relaxation of tick control in North Rift, Kenya.

A total of 90 animals was immunized against East Coast fever (ECF) using Theileria parva (Marikebuni) stock on three large-scale farms in Kiminini Division, Trans-Nzoia District, North Rift, Kenya. Another 90 cattle served as non-immunized controls. Following immunization the number of cattle with significant indirect fluorescent antibody (IFA) titres increased from 43.9% to 84.4% and 6.7% of the cattle developed clinical ECF reactions. Two months after immunization, the immunized and non-immunized cattle were divided into two groups one of which was dipped every 3 weeks and the other dipped when total full body tick counts reached 100. All the animals were monitored for 51 weeks for incidences of ECF and other tick-borne diseases. Twenty-four cases of ECF were diagnosed among the non-immunized cattle compared to four cases among the immunized cattle; a difference that was significant (P > 0.05). There was no significant difference in the incidences of babesiosis and anaplasmosis between the immunized and non-immunized cattle.     

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.     

Comparison of indirect fluorescent antibody test and enzyme linked immunosorbent assay in the detection of exposure of cattle to Theileria parva in Kenya.

Appraisal of the indirect fluorescent antibody test (IFAT) and antigen enzyme linked immunosorbent assay (ELISA) serological tests as carried out to detect cattle exposed to Theileria parva at the National Veterinary Research Centre, Muguga (NVRC), Kenya is reported. Using sera from T. parva naive cattle and cattle experimentally exposed to T. parva, the two tests were appraised in terms of their sensitivity and specificity. IFAT and ELISA had the same sensitivity of 90% while ELISA had a higher specificity (90%) than IFAT (80%). A comparison was also made of the capability of the two tests to detect exposure of dairy cattle to T. parva prior to immunization against East Coast fever (ECF). The positive outcome from the IFAT was significantly higher (chi 2 = 30.36; P < 0.001) than that from the ELISA. The agreement between the two tests was low (Kappa = 0.21). The two tests indicated a higher risk of ECF in the study area than was expected. Indications are that the ELISA has been effectively adopted at NVRC.     

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.     

Epidemiological observations on theileriosis following field immunisation using infection and treatment

Thirty-seven high grade cattle were immunised against Corridor disease (Theileria parva lawrencei infection) on a farm with a history of heavy and often lethal theilerial challenge. Nineteen cattle were immunised by treating with two doses of long-acting oxytetracyclines given at 20 mg/kg on days 0 and 4 after sporozoite stabilate inoculation, while the other 18 were treated with naphthoquinone buparvaquone, given as a single dose of 2.5 mg/kg simultaneously with stabilate inoculation. All the cattle underwent subclinical theilerial reactions with all but two developing high antibody titres on the IFAT test against T. parva schizont antigen by day 35 after the immunisation. Both buparvaquone and long-acting oxytetracycline appeared equally effective in the immunisation. To date, 26 months later, only two cases of theileriosis parasitologically characteristic of T. p. parva have been reported in the immunised cattle. Following the two cases, investigations showed that when uninfected Rhipicephalus appendiculatus nymphal ticks were deliberately fed on healthy resident cattle on the farm, the resultant adult ticks transmitted acute and lethal theilerial infections to five out of five susceptible cattle. The resultant infections were parasitologically characteristic of T. p. parva infections. Furthermore, the monoclonal antibody profiles of schizont infected cell lines from these infections appeared to be characteristic of T. p. parva. It was thus concluded that resident cattle on the farm could be a potential source of T.p. parva infection which had broken through the immunity of T.p. lawrencei immunised cattle and could constitute a reservoir of theilerial infection for ticks and hence to susceptible stock on the farm.     

Orientation of bovine CTL responses towards PIM, an antibody-inducing surface molecule of Theileria parva, by DNA subunit immunization

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8(+) cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.     

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated.     

Analyses of genes encoding Theileria parva p104 and polymorphic immunodominant molecule (PIM) reveal evidence of the presence of cattle-type alleles in the South African T. parva population

Restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) and sequencing of the variable region of the p104 and PIM genes was performed on samples obtained from South African T. parva parasites originating from cattle on farms with suspected theileriosis and from buffalo. p104 and PIM PCR-RFLP profiles similar to those of the T. parva Muguga stock, an isolate that causes ECF in Kenya, were obtained from three of seven cattle samples collected on a farm near Ladysmith in KwaZulu-Natal Province. Amino acid sequences of the p104 and PIM genes from two of these samples were almost identical to the T. parva Muguga p104 and PIM sequences. This result supports findings from a recent p67 study in which p67 alleles similar to those of the T. parva Muguga stock were identified from the same samples. While these results suggest the presence of a cattle-derived T. parva parasite, reports of cattle-to-cattle transmission could not be substantiated and ECF was not diagnosed on this farm. Although extensive diversity of p104 and PIM gene sequences from South African T. parva isolates was demonstrated, no sequences identical to known cattle-type p104 and PIM alleles were identified from any of the buffalo T. parva samples analyzed. 'Mixed' PIM alleles containing both cattle- and buffalo-type amino acid motifs were identified for the first time, and there appeared to be selection of cattle-type and 'mixed'-type PIM sequences in the cattle samples examined.     

Molecular and immunological characterisation of Theileria parva stocks which are components of the 'Muguga cocktail' used for vaccination against East Coast fever in cattle

The 'Muguga cocktail' which is composed of three Theileria parva stocks Muguga, Kiambu 5 and Serengeti-transformed has been used extensively for live vaccination against East Coast fever in cattle in eastern, central and southern Africa. Herein we describe the molecular characterisation of the T. parva vaccine stocks using three techniques, an indirect fluorescent antibody test with a panel of anti-schizont monoclonal antibodies (MAb), Southern blotting with four T. parva repetitive DNA probes and polymerase chain reaction (PCR)-based assays detecting polymorphism within four single copy loci encoding antigen genes. The Muguga and Serengeti-transformed stocks exhibited no obvious differences in their reactivity with the panel of MAbs, whereas Kiambu 5 differed with several MAbs. Kiambu 5 DNA was very distinct from the Muguga and Serengeti-transformed isolates in the hybridisation pattern with all four nucleic acid probes, whereas Muguga and Serengeti-transformed isolates exhibited minor differences and could not be discriminated with one of the probes. PCR amplification in combination with restriction fragment length polymorphism analysis indicated that Kiambu 5 was also markedly divergent from the Muguga and Serengeti-transformed stocks within two of the four antigen coding genes. The T. parva Serengeti-transformed stock did not contain a 130 base pair insert within the p67 sporozoite antigen gene, which has been observed previously in most T. parva parasites isolated from buffalo, and could not be discriminated from T. parva Muguga at any of the four single copy loci. Collectively the data indicate that two of the cocktail components T. parva Serengeti-transformed and Muguga are genetically closely related, while the third component Kiambu 5 is quite distinct. Based on the findings, there may be a need to include only one of the T. parva Muguga and Serengeti-transformed components in the immunising cocktail. The study demonstrates the value of molecular characterisation data for monitoring of live vaccines.     

The effect of East Coast fever immunisation and different acaricidal treatments on the productivity of beef cattle

A trial was performed on a farm in the Coast Province of Kenya to study the effects of East Coast fever immunisation and different acaricidal treatments on the productivity of immunised and unimmunised beef cattle. Eighty cattle were immunised against Theileria parva parva (Marikebuni) by the infection and treatment method and a similar group was left as an unimmunised control. Immunisation had no deleterious effect on the cattle. After immunisation, the immunised and control groups were each subdivided into four groups of 20 and each subgroup was managed under a different tick control regimen. The tick control regimen were, acaricidal spraying twice a week or once every three weeks, the application of acaricide-impregnated ear-tags, and no tick control. During a nine-month exposure period there were 18 cases of East Coast fever among the 80 immunised cattle, three which were severe and the others mild. Among the 80 unimmunised cattle there were 57 cases of East Coast fever, 50 of which were severe. The highest morbidity and mortality occurred in the groups under limited tick control or without tick control. Overall weight gain in the immunised cattle, irrespective of the tick control regimen, was better than the weight gain in the unimmunised groups. Within the immunised groups, the weight gain of the cattle sprayed twice weekly was comparable to the weight gain of the animals with acaricidal ear-tags and was significantly higher than the weight gains in the groups sprayed once every three weeks or with tick control. Preliminary cost/benefit analysis showed that it was uneconomical to maintain unimmunised cattle under limited or no tick control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exposure of cattle immunized with different stocks of Theileria parva to buffalo-associated Theileria challenge on two game parks in Zimbabwe.

Eight cattle immunized with cattle-derived Theileria parva Boleni stabilate together with six susceptible controls were released in Dombawera Game Park on the Highveld of Zimbabwe. This coincided with Rhipicephalus appendiculatus nymphal activity. The cattle grazed together with African buffaloes (Syncerus caffer) and were not treated against tick infestation. The nymphal tick infestation was high, and seven of the eight immunized cattle and three of the controls had severe and fatal reactions. Subsequently, two stocks of Theileria parva to be tested for their immunizing abilities were prepared-one from adult ticks which were fed as nymphs on one of the sick control animals (Dom 268) and the other from adult ticks collected from pastures grazed by buffaloes (Bv-1). Two groups of cattle were immunized with either the Dom 268-derived strain (eight animals) or the Bv-1-derived strain (four animals). These together with three non-immunized controls, were released in Bally Vaughaun Game Park in the Highveld, where buffaloes are present, during the season of nymphal tick activity. A third group of five cattle, immunized with stabilate Bv-1, and three non-immunized controls were released at the same site during the season of adult tick activity. The nymphal and adult tick infestations of the cattle were large and more than 2000 nymphs and 1000 adult ticks were counted per animal. Cattle were treated with a pyrethroid pour-on preparation to control the tick infestation and screw-worm strike. The immunized cattle in the three groups survived the theileriosis challenge for a period of 18 months, but the non-immunized control cattle suffered a severe and fatal theileriosis 19-23 days after being placed on the pasture.     

Theileria parva: the immune status of calves born of dams immunised against East Coast fever

Eight Friesian cross cows three months pregnant to a single Friesian bull were immunised against East Coast fever by infection with Theileria parva (Muguga) sporozoite stabilate and treatment with pyrrolidino-methyl tetracycline. They were challenged with the homologous stock four times before calving and a fifth time after calving, and resisted all five challenges which killed all of the five groups of five susceptible controls. Calves born to these hyperimmunised dams were fully susceptible on challenge with the same stabilate, as were susceptible cows from the same farm and their calves. In both instances the calves died three to seven days earlier than the cows which were approximately 10 times heavier. These results show that one- to two-month-old taurine calves from artificially immunised dams are not protected from experimental T parva sporozoite challenge and that there is no inherent calfhood resistance to East Coast fever.     

Activity of 10 naphthoquinones, including parvaquone (993C) and menoctone, in cattle artificially infected with Theileria parva

Ten naphthoquinones, including parvaquone (993C) (Clexon; Wellcome) and menoctone, were tested for activity in cattle artificially infected with Theileria parva, the causative organism of East Coast fever (ECF). Parvaquone cured all 14 cattle treated with a single dose of 20 mg/kg intramuscularly and all five treated twice with 10 mg/kg intramuscularly. Menoctone cured seven of 10 cattle treated with a single dose of 5 mg/kg intramuscularly. Of 25 untreated control cattle, 22 died of ECF. None of the remaining eight naphthoquinones was as active as parvaquone. Three esters of active naphthoquinones, designed as 'prodrugs' of their parent compounds, showed little or no activity in infected cattle despite being highly active in vitro against T parva. These results were instrumental in the selection of parvaquone for development as the first specifically active remedy for ECF.     

East coast fever: 1. Chemoprophylactic immunization of cattle against Theileria parva (Muguga) and five theilerial strains

In one experiment, groups of cattle were immunized by chemophylaxis or therapy against Theileria parva (Muguga), and were challenged with five recently-isolated strains of Theileria. Two of the strains caused mild or inapparent reactions in the immunized animals, and three caused significant reactions. None of 25 immunized animals died on challenge, whereas 22 of 25 susceptible (non-immunized) animals died.In a second experiment, groups of cattle were immunized against five field strains using chemoprophylaxis. They had minimal reactions and were solidly immune both to isologous challenge and to subsequent challenge with T. parva (Muguga). This demonstrated that cattle could be effectively immunized against lethal challenge with several theilerial strains by inoculating them with 1 ml of an infective stabilate of the appropriate strain and the concomitant daily inoculation of 5 mg/kg of a formulation of oxytetracycline on 4 consecutive days starting on the day of infection.It is suggested that T. parva (Muguga) may be a valuable component of a polyvalent East Coast fever (ECF) vaccine in East Africa.     

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.