Characterisation of a cluster of genes encoding Theileria annulata AT hook DNA-binding proteins and evidence for localisation to the host cell nucleus.

Infection of bovine leukocytes by the apicomplexan parasite Theileria annulata results in alteration of host cell gene expression and stimulation of host cell proliferation. At present, the parasite-derived factors involved in these processes are unknown. Recently, we described the characterisation of a parasite gene (TashAT2), whose polypeptide product bears AT hook DNA-binding motifs and may be transported from the parasite to the host nucleus. We now describe the isolation of a further two genes (TashAT1 and TashAT3) that are very closely related to TashAT2. All three TashAT genes are located together in a tight cluster, interspersed by two further small open reading frames, all facing head to tail. TashAT2 was shown to be expressed in all T. annulata cell lines examined, whereas TashAT1 and TashAT3 were expressed in the sporozoite stage of the parasite, and also in infected cell lines, where their expression was found to vary between different cell lines. Evidence for transport was provided by antisera raised against TashAT1 and TashAT3 that reacted with the host nucleus of T. annulata-infected cells. Reactivity was particularly strong against the host nuclei of the T. annulata-infected cloned cell line D7B12, which is attenuated for differentiation. A polypeptide in the size range predicted for TashAT3 was preferentially detected in host enriched D7B12 nuclear extracts. DNA-binding analysis demonstrated that fusion proteins containing the AT hook region of either TashAT1 or TashAT2 bound preferentially to AT rich DNA.

Temporal co-ordination of macroschizont and merozoite gene expression during stage differentiation of Theileria annulata.

The bovine parasite, Theileria annulata has a complex life-cycle involving the expression and repression of genes during development of its morphologically distinct life-cycle stages. In order to detail the molecular events that occur during differentiation of the intracellular multinucleate macroschizont to the extra-cellular uninucleate merozoite, we have isolated two genes, Tash1 and Tash2 which are differentially expressed during differentiation. Nuclear run on data show that Tash1 gene expression is controlled, at least in part, at the level of transcription. Immunofluorescence data identify the macroschizont as the location for both Tash1 and Tash2 gene products. Northern blot analysis of these genes indicated that their mRNA levels decrease during differentiation in vitro, at a time point coincident with major elevation in the mRNA levels of the merozoite antigen, Tams1, shown previously to be associated with commitment to merozoite production. Furthermore, experiments where cultures were incubated at 41 degrees C for 4 days and replaced at 37 degrees C for 2 days demonstrated that re-expression of Tash1 occurred and is probably linked to reversion to the macroschizont and decreased expression of Tams1. These results imply that the control of macroschizont and merozoite gene expression during differentiation is closely co-ordinated temporally. In addition, a comparison of Tash2 and Tams1 expression has indicated that translational or post-translational control of gene expression may operate in the undifferentiated macroschizont.

Failed venture: Gordon Murray and the W. P. Caven Memorial Research Foundation, 1949-74.

This paper examines the founding and operation of the W. P. Caven Memorial Research Foundation, a private Toronto laboratory, which existed from 1949 to 1974. The Caven Foundation's Research Director was celebrated Toronto surgeon Gordon Murray (1894-1976), who, in 1949, accepted this position amidst personal and public expectations of great medical discoveries and innovations to come. For 25 years Murray carried on his research at the Caven Foundation, generating more controversy and disappointment than medical cures, before the laboratory was closed for financial reasons. What might have been a successful alternative to the University-based medical research structure in Canada resulted in a failed venture. The Foundation did not become a viable research centre largely because of its Research Director and his inability to adapt to the many changes occurring in the conduct and funding of clinical research. The history of the Caven Foundation is explored here within the context of increasingly specialized research techniques and methodology, the rising predominance of the interdisciplinary research team, and the new system of grantsmanship.

An upstream element of the TamS1 gene is a site of DNA-protein interactions during differentiation to the merozoite in Theileria annulata.

Apicomplexan parasites are major pathogens of humans and domesticated animals. A fundamental aspect of apicomplexan biology, which may provide novel molecular targets for parasite control, is the regulation of stage differentiation. Studies carried out on Theileria annulata, a bovine apicomplexan parasite, have provided evidence that a stochastic process controls differentiation from the macroschizont to the merozoite stage. It was postulated that this process involves the presence of regulators of merozoite gene expression in the preceding stage of the life cycle, and that during differentiation a quantitative increase of these factors occurs. This study was carried out to test these postulations. Nuclear run-on analysis showed that TamS1 expression is controlled, at least in part, at the transcriptional level. The transcription start site showed homology with the consensus eukaryotic initiator motif, and study of the 5' upstream region by the electrophoretic mobility-shift assay demonstrated that a 23 bp motif specifically bound factors from parasite-enriched nuclear extracts. Three complexes were shown to bind to a 9 bp core binding site (5'-TTTGTAGGG-3'). Two of these complexes were present in macroschizont extracts but were found at elevated levels during differentiation. Both complexes contain a polypeptide of the same molecular mass and may be related via the formation of homodimer or heterodimer complexes. The third complex appears to be distinct and was detected at time points associated with the transition to high level merozoite gene expression.

Treatment of infected left ventricular assist device using antibiotic-impregnated beads.

There is no well-established therapy for treating infections of heart-assist or artificial heart devices, a serious problem with life-threatening consequences. We used a promising new approach in which antibiotic-impregnated polymethylmethacrylate beads were placed around an implanted left ventricular assist device to control an external blood pump infection in a bridge-to-transplant patient. In this case report, we describe the potential of antimicrobial-impregnated polymethylmethacrylate beads for in situ control of infections involving external surfaces of cardiovascular devices.

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.

Take Five, a nutrition education intervention to increase fruit and vegetable intakes: impact on consumer choice and nutrient intakes.

This study reports results from a randomized controlled intervention trial, focusing on: (1) the identification of successful consumer strategies for increasing fruit and vegetable intakes to the recommended levels of more than five (80 g) portions per day and (2) impact on overall diet and nutrient intakes. Adult men and women (n 170) fulfilling the main recruitment criterion of eating less than five fruit and vegetable portions per day but contemplating increasing intakes were recruited. Complete valid dietary data was provided by 101 intervention (fifty-nine estimated fruit and vegetable intakes, and forty-two simultaneous weighted total dietary and estimated fruit and vegetable intakes) and twenty-four control subjects (weighed total dietary intakes). Intervention advice included the specific association of high fruit and vegetable intake with reduced risk of disease, practicalities, and portion definition with a target intake of greater than five 80 g fruit and vegetable portions per day for 8 weeks. There were significant effects (P < 0.001) on weighed intakes of fruit and vegetables in the intervention group, rising from 324 (SE 25) to 557 (SE 31) g/d and reflected by validated portion measures at 8 weeks intervention. Successful strategies chosen by 'achievers' of the target intake (65% of subjects) were conventional (fruit as a snack, vegetables with main meals etc.) and favoured fruit. There were significant increases in percentage energy from carbohydrate (from sugars not starch), vitamin C, carotenes and NSP and there was a significant decrease in percentage energy from fat for subjects who had high fat intakes (> 35% energy) at baseline. Follow-up self-reported measures at 6 and 12 months indicated mean intakes of 4.5 and 4.6 defined portions/d respectively, suggesting some sustainable effect. In conclusion, the intervention led to significant increases in fruit and vegetable intakes largely via conventional eating habits, with some desirable effects on macro- and micronutrient intakes.

Take Five, a nutrition education intervention to increase fruit and vegetable intakes: impact on attitudes towards dietary change.

To assess the response of low consumers of fruit and vegetables to a nutrition education intervention programme, data were collected from 104 adults on attitudinal variables related to 'eating more fruit, vegetables and vegetable dishes'. Questionnaires (based on the theory of planned behaviour) assessing perceived barriers to increasing fruit and vegetable consumption were administered before an action-orientated intervention programme and at the end of the intervention period (8 weeks). Questionnaire scores for belief-evaluations in the intervention groups pre- and post-study indicated that support of family and friends, food costs, time constraints and shopping practicalities (in order to increase intake of fruit, vegetable and vegetable dishes) were barriers to greater consumption of these foodstuffs. Perceived situational barriers to increasing intakes of fruits and vegetables were: limited availability of vegetables, salads and fruit at work canteens, take-aways, friends' houses and at work generally. Following the intervention the number of visits to the shops was perceived as a greater barrier for increasing intakes of fruit and vegetables. Perceived practical opportunities for increasing intakes high-lighted drinking fruit juice, taking fruit as a dessert, having fruit as a between-meal snack and eating two portions of vegetables with a meal. About two-thirds of intervention subjects achieved the recommended fruit and vegetable target, but it is concluded that practical issues and situational barriers need to be addressed for the success of future public health campaigns.

Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests.

The divergence of parasites is important for maintenance within an established host and spread to novel host species. In this paper we have carried out phylogenetic analyses of Theileria parasites isolated from different host species. This was performed with small subunit ribosomal RNA sequences available in the data bases and a novel sequence amplified from Theileria lestoquardi DNA. Similar phylogenetic studies were carried out with sequences representing the major merozoite/piroplasm surface antigen (mMPSA) from the data base, and novel sequences representing 2 mMPSA alleles from T. lestoquardi, a full length sequence of a Theileria taurotragi mMPSA gene and partial sequences of two new allelic variants of the Babesia equi mMPSA gene homologue. The analysis indicated that the pathogenic sheep parasite T. lestoquardi has most probably evolved from a common ancestor of T. annulata. Interestingly, the level of mMPSA sequence diversity found for T. lestoquardi was surprisingly low, while diversity between the B. equi sequences was higher than that found within any of the classical Theileria species. The possible implications of these results for the establishment of Theileria parasites within novel species are discussed. Extensive cross-reactivity of a range of antisera was found when tested against recombinant mMPSA polypeptides from different Theileria (including B. equi) species. The cross-reactivity between mMPSA polypeptides and sequence diversity are relevant for the development of species specific diagnostic tests.

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.

Modulation of protein synthesis relative to DNA synthesis alters the timing of differentiation in the protozoan parasite Theileria annulata.

The control of differentiation through time is critical for the correct ordering of sequential developmental events. A timing mechanism based on the number of mitotic divisions has been proposed for both higher eukaryote and protozoan parasite cellular differentiation. However, the mitotic clock model has not been validated by experiments which altered the proliferation rate of cells in vitro. This study has used the drugs aphidicolin and oxytetracycline to investigate the modulation of differentiation in the protozoan parasite Theileria annulata. The results showed that the timing of macroschizont to merozoite differentiation correlated with expression levels of a merozoite surface antigen during the reversible phase of the differentiation process. In addition, analysis of the effect of the drugs and elevation of culture temperature indicated that altered timing of differentiation was associated with changes to the rate of protein synthesis relative to DNA synthesis. From these results we postulate that the differentiation clock runs on the basis of a progressive elevation of a regulator(s) of merozoite gene expression to a quantitative commitment threshold. We also propose that this mechanism of timing can be corrupted by modulation of the proliferation potential (DNA synthesis) and/or growth potential (factor production) of the cell. The relevance of this model to differentiation in vivo and to other eukaryotic systems is discussed.

Measuring fruit and vegetable intake: is five-a-day enough?

OBJECTIVE: Validation of a self-monitoring "portions' measurement of fruit and vegetable (F&V) consumption against a standard of weighed intakes. DESIGN: Component of a randomized controlled trial. SETTING: Subjects attended research centres in Reading and Glasgow for instruction and monitoring but undertook free-living dietary changes at home. SUBJECTS: A study sample of 42 adult men and women fulfilling the main recruitment criterion of eating less than five F&V portions/day but contemplating increasing intakes and providing weighted baseline reported energy intakes exceeding (estimated basal metabolic rate x 1.1). INTERVENTIONS: Subjects attended an intensive group advice session which included the specific relationship of high F&V intake with reduced risk of disease; practicalities; portion definition and measurement recording. The target was to exceed five F&V portions/day for 8 weeks. MAIN OUTCOME MEASURES: Self-recorded simultaneous weighed inventories and F&V portion measures. RESULTS: Data from subjects who were not evident under-recorders showed correlations between portion and weighed intakes of r = 0.73, (P < 0.000), although the portions measure tended to under-estimate intakes. Using 80 g/portion the "5-a-day' concept tends to create false negatives (namely consumption could be greater than 400 g whilst recording fewer than five discrete portions) but rarely false positives (namely recorded consumption of less than 400 g did not give measures of more than five discrete portions). CONCLUSIONS: The data suggest that the five portions F&V/day health message, if used in conjunction with defined discrete portions, would encourage desirable consumption exceeding 400 g.

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.

Disruption of synchrony between parasite growth and host cell division is a determinant of differentiation to the merozoite in Theileria annulata.

The multinucleated macroschizont stage of the protozoon Theileria annulata is an intracellular parasite of bovine leukocytes. The parasite induces the host cell to proliferate, and divides in synchrony with the immortalised host cell. Differentiation to the next stage occurs within the host cell culminating in the release of merozoites and destruction of the leukocyte. In this study clones of Theileria annulata macroschizont-infected cell lines were isolated by limiting dilution and tested for differentiation to the merozoite stage (merogony). Two cloned cell lines underwent differentiation with enhanced efficiency, while two others were of lower efficiency. Quantification was carried out using monoclonal antibodies, which showed that over 90% of the cells in an enhanced cloned cell line could be induced to differentiate. By carrying out induction at 41 degrees C for limited periods of time followed by culture at 37 degrees C evidence was obtained that differentiation to the merozoite is a two-step process: a preliminary reversible phase, followed by a second irreversible phase of differentiation. Analysis of the nuclear number of the macroschizont and the growth rate of the cloned cell lines showed that the ability to differentiate was associated with an increase in nuclear number (size) of the macroschizont, generated by a disruption in the synchrony between parasite growth and host cell division. We believe that these results reveal a relationship between a reduction in parasite division and differentiation, and that there are similarities between stage differentiation in parasites and cellular differentiation in higher eukaryotes.

Characterisation of an extrachromosomal DNA element from Theileria annulata.

Extrachromosomal nucleic acid elements are found in all organisms, commonly as organelle, viral or plasmid genomes. In this paper we describe the initial characterisation of a novel 6.5-kb linear, double-stranded extrachromosomal element from Theileria annulata, and a 2.6-kb RNA species. The DNA element is present in different stages of the life cycle and in different stocks of the parasite. Northern blots of total RNA isolated from different stages of the parasite, probed with the purified element, detect three major transcripts, of 1.45, 1.05 and 0.24 kb, present in all life-cycle stages examined. The possible origin and function of this element is discussed, together with its possible use as a transfection vector for the introduction of genes into protozoan cells.

On the possibility of bovine Onchocerca species infecting Simulium damnosum s.l. in the forest zone of Sierra Leone. II. Biting densities and filarial infections in Simulium spp. and Culicoides spp.

Previous studies of bovine Onchocerca spp. in cattle in Sierra Leone indicated that only O. gutturosa was transmitted in the forest zone at high intensity. To determine its vector(s) and the extent to which Onchocerca-like infections in Simulium damnosum were likely to be of bovine origin, three lines of investigation were pursued. Firstly, a study was made of the biting flies attacking an ox bait animal over a 14-month period at Njala University Campus, near Bo. Secondly, attempts were made to infect the dominant local forest cytospecies of S. damnosum s.l. with O. gutturosa by feeding them on an infected ox under a bed-net. Thirdly, S. damnosum s.l. were infected by intra-thoracic injection of O. gutturosa microfilariae (mff). In 113 collections made at dawn and dusk at weekly intervals from the ox bait, 624 simuliids and 7740 Culicoides spp. were collected. Almost all the simuliids were S. damnosum s.l. which, on the basis of iso-enzyme examination and knowledge of local breeding sites, were identified as S. soubrense 'B'. Although this cytospecies fed readily on the ox at ventral sites where O. gutturosa mff occurred and the bed-net experiments showed that 16.1% of engorged S. soubrense 'B' ingested an average of 3.3 O. gutturosa mff each, no development occurred. The refractoriness of S. damnosum s.l. to O. gutturosa was confirmed by the intra-thoracic injection experiments. The predominant Culicoides spp. attacking the ox bait were C. krameri, C. trifasciellus and C. fulvithorax, with smaller numbers of C. schultzei. In 5803 dissected Culicoides spp., natural infections of Onchocerca-like larvae, presumed to be O. gutturosa, were found in 0.3% of C. fulvithorax, 0.1% of C. trifasciellus and 0.06% of C. krameri. It was concluded that, in the forest zone of Sierra Leone, S. damnosum s.l. is not a vector of O. gutturosa and all Onchocerca-like larvae in S. damnosum are likely to be O. volvulus, while the natural vectors of O. gutturosa are the Culicoides species C. fulvithorax, C. trifasciellus and C. krameri.

Eimeria tenella: local antibodies and interactions with the sporozoite surface.

Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 10(3) oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 10(5) oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and immunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results.(ABSTRACT TRUNCATED AT 250 WORDS)

Class-specific circulating antibodies in infections with Eimeria tenella.

Using a three-layer immunofluorescent test, class-specific, parasite-specific circulating antibody responses to Eimeria tenella were investigated following oocyst infection, drug-truncated oocyst infection and the injection of non-living antigens. Following all three means of antigenic stimulation, IgG, IgM and IgA antibodies were detected. The response to drug-truncated infections was dose-dependent. The sequence of appearance of antibody was IgM, IgA, IgG, whilst the relative quantities were IgG greater than or equal to IgM greater than IgA.

The maintenance and survival of Theileria annulata in colonies of Hyalomma anatolicum anatolicum.

Procedures are described for the colonization of Hyalomma anatolicum anatolicum ticks using gerbils, rabbits and cattle as hosts. On rabbits H. a. anatolicum undergoes a two-host cycle but methods are described for obtaining either unfed or engorged nymphs. Data are given on the life cycle timings and the numbers and timings of tick feeding regimes. The infection of H. a. anatolicum with two strains of Theileria annulata is described and methods given for monitoring the development and survival of T. annulata in H. a. anatolicum. Data are presented indicating the optimum maintenance conditions for T. annulata in H. a. anatolicum. Piroplasm parasitaemias in cattle greater than 2% gave high infection rates in adult ticks if engorged nymphs were moulted at 28 degrees C for 28 days. The Theileria will survive for ten months in such adults stored at 12 degrees C after moult, and when fed the ticks will produce maximum numbers of sporozoites on the third day of feeding, whatever their age. The moult of engorged nymphs is retarded at 18 degrees C but the Theileria in such ticks will develop normally when the ticks are moulted at 28 degrees C after four months storage at 18 degrees C.