The FIKK kinase of Toxoplasma gondii is not essential for the parasite's lytic cycle.

FIKK kinases are a novel family of kinases unique to the Apicomplexa. While most apicomplexans encode a single FIKK kinase, Plasmodium falciparum expresses 21 and piroplasms do not encode a FIKK kinase. FIKK kinases share a conserved C-terminal catalytic domain, but the N-terminal region is highly variable and contains no known functional domains. To date, FIKK kinases have been primarily studied in P. falciparum and Plasmodium berghei. Those that have been studied are exported from the parasite and associate with diverse locations in the infected erythrocyte cytosol or membrane. Deletion of individual P. falciparum FIKK kinases indicates that they may play a role in modification of the infected erythrocyte. The current study characterises the single FIKK gene in Toxoplasma gondii to evaluate the importance of the FIKK kinase in an apicomplexan that has a single FIKK kinase. The TgFIKK gene encoded a protein of approximately 280kDa. Endogenous tagging of the FIKK protein with Yellow Fluorescent Protein showed that the FIKK protein exclusively localised to the posterior end of tachyzoites. A Yellow Fluorescent Protein-tagged FIKK and a Ty-tagged FIKK both co-localised with T. gondii membrane occupation and recognition nexus protein to the basal complex and were localised apical to inner membrane complex protein-5 and Centrin2. Deletion of TgFIKK, surprisingly, had no detectable effect on the parasite's lytic cycle in vitro in human fibroblast cells or in acute virulence in vivo. Thus, our results clearly show that while the FIKK kinase is expressed in tachyzoites, it is not essential for the lytic cycle of T. gondii.

Generation of a mosaic pattern of diversity in the major merozoite-piroplasm surface antigen of Theileria annulata.

The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of the protozoan parasite Theileria annulata. Generation and selection of divergent antigenic types has implications for the inclusion of the Tams1 antigen in a subunit recombinant vaccine or use in the development of a diagnostic ELISA. In this study a total of 129 Tams1 sequences from parasites isolated in Bahrain, India, Italy, Mauritania, Portugal, Spain, Sudan, Tunisia and Turkey were obtained to estimate the extent of Tams1 diversity throughout a wide geographical range. Significant sequence diversity was found both within and between isolates and many of the sequences were unique. No geographical specificity of sequence types was observed and almost identical sequences occurred in different geographical areas and a panmictic population structure is suggested by our results. A sliding window analysis identified sub-regions of the molecule where selection for amino acid changes may operate. Evidence is also presented for the generation of diversity through intragenic recombination with switching of corresponding variable domains between alleles. Recombination to exchange variable domains appears to occur throughout the length of the gene sequence, and has the potential to generate a mosaic pattern of diversity.

Molecular characterisation of the Theileria buffeli/orientalis group.

Benign bovine Theileria parasites known as either Theileria buffeli, Theileria orientalis or Theileria sergenti are classified on basis of their morphology, vector specificity, pathogenicity and 18S small subunit ribosomal RNA or major piroplasm protein (MPSP) sequences. Since most isolates have been characterized on only some of these criteria and the existing confusion in nomenclature, an analysis was performed on eight different isolates to combine 18S rRNA data with MPSP data and the results were compared with available biological parameters. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach for both genes was used in combination with reverse line blot hybridisation for the 18S rRNA gene. Both MPSP and 18S rRNA genes were cloned and sequenced from parasites displaying aberrant MPSP RFLP profiles. Phylogeny based on published and determined 18S rRNA and MPSP sequences did correlate within the same isolate but there was no obvious correlation between molecular and biological data. Based on these findings, we suggest that the appropriate name for all these parasites is Theileria buffeli. A more specific nomenclature should be assigned when new molecular markers may become available.

Characterization of attenuated Theileria annulata vaccines from Spain and the Sudan.

Theileriosis caused by Theileria annulata can be effectively prevented by vaccination with attenuated, cultured schizonts. Although these attenuated vaccines have been applied for a long time, not much is known about the fate of the vaccine strain in the field. Here, two experimental Spanish vaccine strains originating in Cádiz and Cáceres, and one Sudanese strain are studied to address the development of a carrier status and the infectivity for Hyalomma ticks. Moreover, the heterogeneity of the merozoite surface protein, Tams1, was analyzed in search for an attenuation marker. Using the sensitive reverse line blot (RLB) hybridization, the development of a low level carrier status was demonstrated in the Cáceres and Sudanese line vaccinated calves. Although no signal was detected in the Cádiz line vaccinated calves, seroconversion against the schizont stage was observed, as it was in all other calves. The experimental transmission of T. annulata by Hyalomma ticks to naïve calves was unsuccessful for all cell line inoculated calves. Tams1 heterogeneity indicated a clonal selection of parasites during the process of attenuation, but the Tams1 sequence itself has no connection with the attenuation status. In conclusion, a carrier status develops in attenuated schizont culture vaccinated calves, but is not infective for Hyalomma ticks. Based on these data, the risk for spread of the vaccine strains in the field may be very low.

Integrated molecular diagnosis of Theileria and Babesia species of cattle in Italy.

A reverse line blot hybridization (RLB) test was developed to specifically identify six Theileria spp. (T. annulata, T. parva, T. mutans, T. velifera, T. taurotragi, and T. buffeli/orientalis) and three Babesia spp. (B. bovis, B. bigemina, and B. divergens). No cross reaction was observed with other livestock pathogens (such as Anaplasma marginale, A. centrale, A. ovis, Cowdria ruminantium, Trypanosoma brucei, T. congolense, and T. vivax). This method was used to test bovine blood samples collected in Sicily in April and November, 1998. Preliminary results indicated that T. annulata and T. buffeli/orientalis were the main species observed in cattle blood. Babesia species represented 1.8% and 23.5% in April and November, respectively.

Different vaccine strategies used to protect against Theileria annulata.

SPAG-1, a sporozoite surface antigen of T. annulata, has previously been shown to elicit partial protection when used, as an hepatitis B core antigen fusion, to immunize cattle. The objective of this study was to try and improve the protective capacity of this antigen by enlisting different vaccine strategies. Cattle were immunized with SPAG-1, as a fusion protein with a His6 tag, either incorporated into ISCOMs, with or without the merozoite antigens TAMS 1-1 and 1-2, or with RWL as adjuvant three times at monthly intervals. Another group of cattle were immunized with p67, the T. parva sporozoite antigen, in RWL to assess whether any cross-protection could be induced. The animals were then challenged with an estimated LD50 of T. annulata sporozoites, and their ability to resist the infection was investigated. Serum responses and T-cell proliferative responses were analyzed throughout the trial. Post-challenge analyses included lymph node biopsies and blood smears to check for the presence of parasites, routine hematological parameters, and observation for clinical manifestations of the disease. The results of this trial will be discussed.

Development of an indirect Tams1 enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle.

An enzyme-linked immunosorbent assay (ELISA) was developed based on a recombinant major Theileria annulata merozoite surface antigen, Tams1. Four different recombinant proteins derived from two different Tams1 alleles, both in two different truncated forms, were tested for their performance in the ELISA. Furthermore, antigen concentration, various buffers, washing protocol, and the choice of anti-total-immunoglobulin G (IgG), anti-IgG1, or anti-IgG2 as second antibody were evaluated. The performance of the resulting ELISA was analyzed by measuring the coefficient of variation (CV). A total of 22 sera were analyzed over the measurement range, resulting in a CV of ca. 10%, whereas 30% variation is the maximum acceptable. The cutoff value was determined by the two-graph receiver operating characteristic (TG-ROC), using the indirect fluorescent antibody test (IFAT) as a reference. It was shown that up to 3 months postinfection (p.i.) IFAT is more sensitive and specific, whereas beyond 3 months p.i. ELISA performed as well as IFAT. The cutoff was determined at maximal sensitivity, based on the TG-ROC after 3 months p.i. Nine calves experimentally infected with four different T. annulata stocks remained positive in the ELISA for at least 1 year p.i. Finally, limited cross-reaction was found only with T. parva antisera, but not with any other Theileria or Babesia species. Since the T. parva endemic area hardly overlaps with T. annulata, the Tams1 ELISA has the potential to become a useful tool in the epidemiology of tropical theileriosis.

Study of Theileria annulata population structure during bovine infection and following transmission to ticks.

Tams1 is the polymorphic immunodominant merozoite-piroplasm surface protein of Theileria annulata. Evidence for selection of divergent forms of Tams1 has been obtained recently. This study was performed to address whether selection takes place during persistent infection of the bovine host or during passage through the Hyalomma tick vector. Four calves were infected with a T. annulata isolate representing multiple parasite genotypes. The development of the parasite population was analysed by denaturing gradient gel electrophoresis (DGGE) using the Tams1 gene as a marker. In addition, the parasitaemia was measured by a semi-quantitative reverse line blot hybridization assay in order to correlate Tams1 variation to changes in parasitaemia. It was found that both parasitaemia and parasite population displayed limited variation during persistent infection. Ticks were allowed to acquire T. annulata during 2 periods of the bovine infection. Tams1 alleles detected in ticks fed during acute infection were identical to the population in the bovine host. However, ticks fed during the carrier status acquired parasites showing a single Tams1 isotype that represented, in several cases, a minor population in the bovine host at the time of infestation. Although only a limited number of ticks were studied, these preliminary data suggest that specific parasite genotypes may be selected during tick transmission from a carrier animal.

Evaluation of recombinant sporozoite antigen SPAG-1 as a vaccine candidate against Theileria annulata by the use of different delivery systems.

The major sporozoite surface antigen of Theileria annulata (SPAG-1) is a candidate for inclusion in a subunit vaccine. In this paper we summarize the results of 4 vaccination experiments using recombinant SPAG-1 expressed in different systems and presented in different adjuvants. The antigen has been presented as either a C terminal 108 amino acid peptide (called SR1) expressed as both beta-galactosidase and hepatitis B core antigen fusions or as a full-length form expressed as a GST fusion with an N terminal His6 tag. We used different adjuvants, namely Freund's, saponin, ISCOMs and a proprietary adjuvant supplied by SmithKline Beecham, which we call SKBA. The data point to the conclusion that SPAG-1 can elicit partial protection and is therefore suitable for inclusion in an eventual multicomponent subunit vaccine.