Directing differentiation in Theileria annulata: old methods and new possibilities for control of apicomplexan parasites.

Apicomplexan parasites are major pathogens of humans and domesticated animals. The ability of these organisms to evade the host immune response and the emergence of drug-resistant parasites indicates a need for the identification of novel control strategies. Ideally, selected targets should be shared by a range of apicomplexans and fundamental to parasite biology. One process of apicomplexan biology which may provide this type of target is the molecular regulation of stage differentiation. This paper has reviewed studies carried out on differentiation of Theileria annulata and has highlighted general similarities with other apicomplexan differentiation steps. Similarities include asynchrony of differentiation, the loss (attenuation) of differentiation potential and an association between reduced proliferation and differentiation. In addition, novel data are presented assessing a possible role for a signal transduction mechanism or a direct involvement of classical heat-shock polypeptides in regulating differentiation of T. annulata in vitro. These studies, and previously published data, have led to the postulation that progression to the next stage of the life-cycle can be predetermined and involves the attainment of a quantitative threshold by regulators of gene expression. A modification of this model takes into account that for certain in-vitro systems, or differentiation steps in vivo, the process has to be initiated by alteration of the extracellular environment. Work which has shown that the time taken to achieve differentiation can be increased or decreased is also outlined. The ability to change the timing of differentiation suggests that the associated regulatory mechanism could be manipulated directly to significantly influence the outcome of an apicomplexan infection. The observation that a number of existing drugs and control strategies may exert their protective effect by altering differentiation potential supports this possibility.

Selection for antigenic diversity of Tams1, the major merozoite antigen of Theileria annulata.

Tams1, the major merozoite/piroplasm surface antigen of Theileria annulata has the potential to be a component of a diagnostic ELISA test and be included in a recombinant subunit vaccine. However, the observation that this antigen displays diversity could constrain these applications. In this paper we have extensively characterized Tams1 diversity at the DNA level, using a PCR/sequencing strategy. Up to 44 alleles have been cloned and sequenced. The comparison of these alleles has identified regions of sequence conservation, variability and hyper-variability. Computer analysis of these alleles has indicated that positive selection may operate on certain regions of Tams1. Expression and Western blot analysis of selected alleles has indicated that sequence diversity is reflected in altered antigenicity and a continuum of relatedness and antibody cross recognition may exist. The possible function of the sequence conservation and polymorphism within Tams1 is discussed in relation to protein structure, host cell invasion and immune evasion.

Epidemiology of tropical theileriosis (Theileria annulata infection of cattle) in an endemic region of Tunisia: characterisation of endemicity states.

A serological survey on tropical theileriosis was conducted on a sample of 54 farms in a region within the semi-arid bioclimatic zone of Tunisia. Screening of cattle sera at a dilution of 1/160 using the indirect immunofluorescent antibody test with the schizont antigen of Theileria annulata, revealed the presence of animals with positive sera in 92.15% of the sampled farms. The exposure of calves to infection in the first season was shown to be significantly lower than in older cattle. Three endemic situations were identified based on the serological profiles of herds and the incidence and age distribution of disease cases. Endemic stability was observed in farms showing a sero-prevalence of 100% in cattle of four theileriosis seasons or more and by the incidence of the highest disease levels in cattle at their second and third theileriosis season. High endemic instability was identified on the basis of low sero-prevalence rates and the occurrence of the highest disease incidence in cattle at fourth theileriosis season or more.

Cattle infestation by Hyalomma ticks and prevalence of Theileria in H. detritum species in Tunisia.

Seventy-four cattle, from three farms endemic for tropical theileriosis in the north of Tunisia, were studied for tick populations from June 1991 to June 1992. Ticks were removed from cattle twice a month in the summer and every month the rest of the year. They were identified and assessed for Theileria infection. A total of 5083 Hyalomma adult ticks were collected and the major species found was H. detritum (84.3%). The activity of this species is limited between June and August with a peak in numbers observed at the end of June and the beginning of July. Amongst the 2356 Hyalomma ticks dissected, no evidence of salivary gland infection was found in either H. m. marginatum or H. a. excavatum. However, 12.4% (277/2230) of H. d. detritum dissected ticks were infected with Theileria species and amongst these, 62% had one to two sporoblasts in their salivary glands (range 1-91). The prevalence, but not the intensity, of infection was greater in females than in male ticks, and the cases of tropical theileriosis followed the peak of infected females. This suggests that female ticks have a more important role in theileriosis transmission than male ticks. A significantly lower number of adult H. detritum were collected from calves, at their first tick season, than from adult cattle. Finally, this study showed that the infestation level of cattle by H. d. detritum and the prevalence of Theileria-infection in these cattle varied between the three farms studied.

A preliminary study on the attenuation of Tunisian schizont-infected cell lines of Theileria annulata.

Four Theileria annulata cell lines were characterised at low passage levels using two polymorphic markers and then used to infect calves. Their virulence seemed to be related to the number of genotypes present within the cell line. In all, 3 of the 4 cell lines were cultured up to passage 100 or 200 and inoculated into calves. Their characterisation using the same markers indicated that the attenuation was related to a reduction in the parasite polymorphism down to a single genotype. The immunogenicity of the three attenuated cell lines was assessed in calves using two types of challenge. Optimal protection was observed against homologous challenges. The level of immunity to heterologous challenges appeared to decrease with attenuation and seemed to depend on the cell line used.

Diagnosis of Theileria annulata infection of cattle in Tunisia: comparison of serology and blood smears.

The immunofluorescent antibody test (IFAT) using schizont and piroplasm antigens was compared with the microscopic examination of Giemsa-stained blood smears for the diagnosis of Theileria annulata infection in experimentally and naturally infected cattle. The results obtained on 100 naive cattle showed that non-specific fluorescence disappeared at serum dilution levels of 1/40 and 1/160 for the piroplasm and schizont antigens, respectively. These levels were therefore retained as the starting dilutions for this study. On day 30 post-infection, 16 experimentally infected calves were shown to be serologically positive for schizont and piroplasm IFAT, while 13 of them were positive for blood smears. A total of 109 cattle from an endemic region of tropical theileriosis were sampled before the onset of the disease season in April and later in September. Globally the IFAT results revealed more cattle exposed to T annulata infection than the blood smear examination. The piroplasm IFAT and the blood smears were less reliable than the schizont IFAT. The latter appeared to be the best test for detecting exposure to T annulata.

Selection of diversity at putative glycosylation sites in the immunodominant merozoite/piroplasm surface antigen of Theileria parasites.

The immunodominant merozoite/piroplasm surface antigen of Theileria parasites has potential as a diagnostic reagent and as a component of a sub-unit vaccine. This molecule is known to be antigenically diverse, and it is important to determine the nature and extent of this heterogeneity. In the present study nucleotide sequences, representing alleles of the gene (Tams1) encoding this molecule in Theileria annulata were compared to each other and to sequences of homologous genes in Theileria sergenti, Theileria buffeli and Theileria parva. This analysis revealed that a region of the polypeptide which contains putative N-linked glycosylation sites is particularly diverse and, in analogy to retroviral systems, may indicate selection of variable glycosylation sites or amino acid epitopes to evade the bovine immune response. This conclusion was also made from the results of a phylogenetic analysis which compared the variable region of the genes with a second region, which appeared to show no bias for diversity or functional constraint. The results indicated that the variable sequence encoding putative glycosylation sites has diverged, both within and between Theileria species, at a much faster rate than the rest of the molecule. Southern blot analysis of T. annulata populations from within a single geographical region detected six possible variant Tams1 alleles. However, a correlation between restriction-fragment-length polymorphism (RFLP) patterns detected by the Tams1-1 gene probe and geographical location could not be made. In addition, although a high prevalence of one particular RFLP was found, this is unlikely to be the result of a clonal population structure, as we present evidence for significant parasite genotypic variability within a single endemic region.

Genomic and phenotypic diversity of Tunisian Theileria annulata isolates.

This study describes polymorphism in Theileria annulata, an intracellular protozoan parasite of bovine leucocytes and red blood cells. Fifty-three different stocks of T. annulata, isolated from 17 sites (districts) in Tunisia, have been characterized by anti-parasite monoclonal antibody (MAb) reactivity, glucose phosphate isomerase (GPI) isoenzyme electrophoresis, and Southern blotting with two genomic DNA probes. These appears to be considerable diversity amongst T. annulata stocks from Tunisia, no two isolates being identical, even those from animals on the same farm. Two distinct antigenic populations were detected by MAb 7E7. They were defined by negative and positive cells in the indirect fluorescent antibody test. The percentage of positive cells in different isolates ranged between 0 and 100%. The population variation seen by GPI analysis and DNA probes was greater; 7 different GPI phenotypes were identified amongst the stocks studied, while DNA probes T. annulata Tunis (TaT) 17 and 21 detected up to 5 different variants. The majority of isolates were shown to contain more than one parasite population, the number of variants per isolate ranging from 1 to 4. No correlation between particular parasite phenotypes or genotypes and their geographical site of isolation was observed. Selection of parasite populations in vivo and in vitro is also discussed.