Biomedical subjects
A Tait
Publications and source records attributed to A Tait.
Polymorphism of SPAG-1, a candidate antigen for inclusion in a sub-unit vaccine against Theileria annulata.
SPAG-1, a Theileria annulata sporozoite surface antigen, is a vaccine candidate. Data is presented, based on the clonal segregation of SPAG-1 associated RFLPs, showing that this antigen is encoded by a single copy gene. We have cloned and sequenced a full-length genomic copy of the SPAG-1 gene and a comparison of this with a previously published SPAG-1 cDNA sequence demonstrates a high degree of polymorphism. We infer that these sequences represent two distinct allelic SPAG-1 variants. The deduced polypeptides show an overall identity of 92% with the most variable stretch (60% identity) occurring towards the middle of the molecule. The N and C termini are more conserved with identities of 92% and 97% respectively. The elastin receptor ligand, VGVAPG, present 3 times in the protein sequence derived from the cDNA is not found in that deduced from the genomic copy. Evidence for 2 further SPAG-1 alleles was obtained from PCR based sequences using macroschizont clones containing different SPAG-1 associated RFLPs. In summary we have shown the existence of at least 4 highly polymorphic SPAG-1 alleles. The implications of such polymorphism between and within distinct geographical isolates for the development of a SPAG-1 based subunit vaccine is discussed.
Surface and excretory/secretory antigens of fourth-stage larvae and adult Ostertagia circumcincta.
With the use of an enzyme-linked immunosorbent assay (ELISA), it was revealed that surface antigens of both adult and fourth-stage larvae (L4) of Ostertagia circumcincta induced high levels of serum IgG antibodies, while serum IgA antibody levels were low but increased significantly (P < 0.01) after infection. Immunofluorescence studies on the surface of viable L4 and adult nematodes showed that the IgG response was stage-specific only in animals vaccinated with adult surface extracts. The results of Western blot analysis using these antibodies suggested that at least eight polypeptides were shed from the L4 surface to the environment and that infection induced (or boosted) IgG antibody against a further four polypeptides. A comparison of reactivity of pre- and post-infection sera of sheep vaccinated with adult nematode surface antigens suggested that only one of the antigens stripped from the nematode surface was immunogenic and/or present in a concentration sufficient to induce an IgG response following parenteral vaccination. Infection boosted the IgG antibodies to a further four polypeptides. Only one polypeptide of 63 kDa seems to be shed in vivo from the adult nematode surface. Ten to eleven antigens were recognised in adult excretory/secretory products by serum IgG of multiple-infected sheep.
Trypanosoma brucei: characterization of protein kinases that are capable of autophosphorylation in vitro.
Autophosphorylation by protein kinases has been implicated as an important control mechanism in signal transduction and growth regulatory pathways in mammalian cells. We have set out to investigate whether any such autophosphorylating protein kinase activities can be found in Trypanosoma brucei. In order to do this, we have developed a system for characterizing such protein kinase activities using an in vitro assay. This assay was carried out by fractionation of trypanosome lysates using isoelectric focusing gel electrophoresis followed by incubation of the gel in gamma 32P-labelled nucleotide triphosphate and subsequent autoradiography. We have identified two classes of autophosphorylating protein kinase activities. In the first class all were dependent on ATP as the phosphate donor substrate and were all found to have a molecular size of 60 kDa. Differences in the activity of these protein kinases were observed between the bloodstream and procyclic life-cycle stages. Furthermore, the addition of mammalian epidermal growth factor to bloodstream stage lysates stimulated an additional activity. The second class of autophosphorylating protein kinases utilized GTP as the phosphate donor and were all found to be 90 kDa in size. Stage-specific differences were also observed in the activity of these protein kinases.
Analysis of a new genetic cross between two East African Trypanosoma brucei clones.
Two clones of East African Trypanosoma brucei, with distinct homozygous isoenzyme patterns for one of three enzymes examined, were cotransmitted through the tsetse fly vector Glossina morsitans centralis. Flies with mature infections were individually fed on mice and the subsequent bloodstream from populations analysed for the presence of hybrid trypanosomes by isoenzyme analysis. Several combinations have previously been detected using this approach (Schweizer, Tait & Jenni, 1988; Sternberg et al. 1989). Four clones were isolated from one of the hybrid-containing populations. They showed a hybrid phenotype, as would be expected for the F1 progeny in a diploid Mendelian system. The analysis of the progeny clones, using two gene probes which detect restriction fragment length polymorphisms between the two parental stocks, showed that alleles had segregated at each locus and given rise to three different non-parental combinations of alleles in the hybrid progeny. Characterization of the hybrid progeny clones by PFGE (pulsed field gradient gel electrophoresis) revealed that all progeny clones were recombinant for the intermediate size chromosomes. From the analysis of the segregation of the larger chromosomes, marked by PGK (phosphoglycerate kinase) and CP (cysteine protease) gene probes, it was inferred that the progeny clones did not result from a direct fusion of diploid cells. Results with the PGK probe fit into a classical system with meiosis and subsequent fusion of the nuclei to form diploid progeny. On the other hand, blots with the CP probe as well as some of the ethidium bromide stained PFGE gels revealed the existence of non-parental size chromosomes in some of the hybrid progeny. This phenomenon was observed previously (Gibson, 1989) and further investigation is required to elucidate the mechanism.
Epidemiological relationships of Trypanosoma brucei stocks from south east Uganda: evidence for different population structures in human infective and non-human infective isolates.
This study represents an analysis of trypanosome strains circulating within a confined location over a short period of time during a sleeping sickness epidemic in S.E. Uganda. A large number of Trypanosoma brucei isolates (88) were collected from a variety of hosts (man, cattle, pigs and tsetse) from villages within a 10 km radius and were analysed for variation in isoenzyme patterns, restriction fragment length polymorphism (RFLP) in repetitive DNA sequences and susceptibility to human serum. The human infective stocks form a clearly distinguishable population when compared with other stocks circulating in the domestic cattle reservoir. The data here support the occurrence of genetic exchange between the cattle stocks while an 'epidemic' population structure involving limited genetic exchange is a characteristic of the human infective stocks. Furthermore, it is shown that when both RFLP and isoenzyme analysis are carried out most stocks appear to have individual genotypes. Stocks which were formerly grouped as zymodemes are better considered as a collected of distinct individuals.
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.
Theileria annulata sporozoite surface antigen (SPAG-1) contains neutralizing determinants in the C terminus.
SPAG-1 is a surface antigen on Theileria annulata sporozoites that is a candidate both for inclusion in a subunit vaccine and as a ligand for host cell recognition. We have pinpointed major neutralizing epitopes to the C terminus. To facilitate this we expressed SPAG-1 as a series of defined fragments in the pGEX system. These constructs were validated by sequencing and by their spectrum of reactivity with monoclonal antibody (MoAb) BA4. This MoAb recognizes the elastin motif VGVAPG, that is predicted to occur three times in the N terminal half of SPAG-1. The recombinant proteins were then tested by Western blotting with a neutralizing MoAb (1A7) and two neutralizing bovine sera (10T and 34A). The results demonstrate that 1A7 and the bovine sera react with determinants unique to the C terminus. We mapped the neutralizing determinant recognized by MoAb 1A7 to a 16 residue sequence (residues 807-822) using synthetic peptides. Interestingly the bovine sera do not recognize the 1A7 epitope. The potential role of the C terminus as a ligand for host cell recognition and the implications for sub-unit vaccine production are discussed.
A novel CDC2-related protein kinase from Leishmania mexicana, LmmCRK1, is post-translationally regulated during the life cycle.
The p34CDC2 protein kinase is a key component in the regulation of the eukaryotic cell cycle. We have isolated from the protozoan parasite Leishmania mexicana mexicana a CDC2-related kinase gene (Lmmcrk1) encoding a 34-kDa protein kinase (lmmCRK1) which has 56% amino acid identity with the human CDC2 and contains a PCTAIR motif in place of the highly conserved PSTAIR box. lmmCRK1 was detected in all life cycle stages at comparable levels, yet its histone H1 kinase activity was detected in only the promastigote form, indicating that its activity is stage-regulated at a post-translational level. lmmCRK1 did not bind p13suc1 beads and Lmmcrk1 was unable to complement a fission yeast temperature-sensitive cdc2 mutant. These data suggest that Lmmcrk1 is unlikely to be the functional L. mexicana cdc2 homologue. A distinct histone H1 kinase activity that binds p13suc1 beads (SBCRK) was also detected, with activity that correlated with the division status of the developmental forms of the parasite, being present in the dividing stages of the parasite and absent in nondividing metacyclic forms. SBCRK is a candidate for the functional CDC2 homologue, but it does not react with an anti-PSTAIR monoclonal antibody on Western blots when eluted from p13suc1 beads, indicating a divergent PSTAIR box. These data suggest that a family of CDC2-related protein kinases are present in Leishmania. Some share sequence and biochemical properties with CDC2, but significant differences also exist, possibly reflecting the evolutionary distance between Leishmania and higher eukaryotes.
Identification of ornithine decarboxylase as a trait gene for growth in replicated mouse lines divergently selected for lean body mass.
Studies of lines of mice selected for body mass have shown that there is a significant genetic component affecting this trait although the nature of the genes involved remains to be elucidated. Using replicate lines of mice, our studies have shown that two different variants of the mouse ornithine decarboxylase (ODCase) gene have been selected in replicate lines of mice selected for high and low lean body mass respectively. One variant is associated with an increased peak of ODCase activity in embryos (10-13 days of gestation) in all high mass lines and with a restriction fragment length polymorphism of the expressed gene. The increased ODCase activity coincides with increased ODCase mRNA levels in the high mass selected lines. These results provide evidence implicating ornithine decarboxylase as a major factor in cell growth, and as a candidate 'trait gene'.
Characterization of Megatrypanum trypanosomes from European Cervidae.
Megatrypanum trypanosomes have been isolated from a number of different European Cervidae, but on the basis of morphology it has not been possible to define the species to which these isolates belong. We isolated Trypanosoma (Megatrypanum) theileri from 10 cattle, and Megatrypanum trypanosomes from 11 fallow deer (Cervus dama), 9 red deer (Cervus elaphus), and 4 roe deer (Capreolus capreolus) by blood culture on a biphasic medium (NNN agar slopes). Trypanosomes were propagated in Schneider's Drosophila medium and characterized by isoenzyme analysis and molecular karyotyping. Isocitrate dehydrogenase and phosphoglucomutase were visualized after starch gel electrophoresis of trypanosome lysates. By cluster analysis of this data all isolates from deer were clearly separated from the T.(M.) theileri isolates from cattle. Isolates from roe deer were different not only from T. (M.) theileri but also from the other deer isolates. Isolates from fallow deer and red deer were grouped together. Thus, there are probably at least two different species of Megatrypanum trypanosomes in the three Cervidae. One parasitizing roe deer, the other, apparently less host specific species, infecting red deer and fallow deer. Separation of the chromosomes of Megatrypanum trypanosomes by pulsed-field gradient gel electrophoresis (PFGE) showed that each isolate contained a large number (> 18) of chromosomes ranging in size from 300 to > 2200 kb. The molecular karyotypes were similar for all isolates, although no isolate was identical to another.
Synthesis and antiinflammatory activity of 2,6-bis(1,1-dimethylethyl)phenol derivatives.
The influence of 11 newly synthesized 2,6-bis(1,1-dimethylethyl)phenol derivatives substituted in the 4 position as measured on the carrageenan paw edema assay in Sprague-Dawley rats, was studied using indomethacin as a reference drug. Furthermore we studied the possible interference of few of these compounds on the calcium binding sites by using the specific ligand [3H]-PN 200-110 in "in vitro" experiments. As far as regard the antiinflammatory activity only the compounds 2b, 2j and 2k, dosed at 20 mg/Kg/os, exerted an inhibitory effect on paw edema which was practically equal, after 6 h, to that of indomethacin (approximately 30%) dosed at 2.5 mg/Kg. The compound 2k, however, showed, in comparison with indomethacin and the other new tested compounds, a longer lasting effect, reaching, after 8 h, a 56.7% inhibition of the edema. Finally the above mentioned compounds, when tested alone or in combination with nitrendipine, did not exert any displacing activity on [3H]-PN 200-110 binding to synaptosomal membranes. It is noteworthy however that compound 2e, which incidentally was inactive as antiinflammatory agent, showed a negative allosteric modulatory activity on the ability of nitrendipine to displace [3H]-PN 200-110 binding.
Theileria annulata: alterations in phosphoprotein and protein kinase activity profiles of infected leukocytes of the bovine host, Bos taurus.
The phosphoprotein profiles of T. annulata-infected and uninfected leukocyte cell lines were compared by both in vivo and in vitro labeling assays. Three phosphoproteins were unique to T. annulata-infected cells, while a further two were quantitatively increased relative to uninfected cells. In addition, two proteins were present and phosphorylated only in the uninfected cell lines, suggesting either a repression of these proteins or their dephosphorylation upon infection of the host cell by the parasite. In order to determine if alterations in protein kinase activity may be responsible for these differences, as opposed to levels of available substrate, an in situ electrophoretic protein kinase assay was developed. This assay allowed a crude separation of protein kinases and revealed alterations in the protein kinase profile of Theileria-infected cells which reflected the differences observed in phosphoprotein profiles. Two protein kinases were unique to infected cells, a further two were more active in infected cells while one was more active in uninfected cells.
Mimicry of elastin repetitive motifs by Theileria annulata sporozoite surface antigen.
Theileria annulata is an important pathogen of cattle in the tropics. The gene sequence of a sporozoite surface antigen (SPAG-1) is reported. Data is also presented demonstrating that SPAG-1 is synthesised as a large precursor. This antigen, which is a candidate for inclusion in a subunit vaccine, shows a remarkable degree of molecular mimicry to the extracellular matrix protein elastin. It contains both repetitive motifs PGVGV and VGVAPG. Immunofluorescence using a monoclonal antibody against VGVAPG confirmed that this peptide is expressed on sporozoites as predicted. The presence of VGVAPG is particularly interesting since this is the ligand for elastin receptors on a range of cell types, including macrophages/monocytes which are a major class of host target cells. It is proposed that this antigen represents the ligand whereby T. annulata recognises its host cells.
Isoenzyme analysis of Haemonchus contortus resistant or susceptible to ivermectin.
Three different strains of Haemonchus contortus (susceptible to ivermectin, S-IVM; selected for resistance to ivermectin, R-IVM; a multiple resistant strain, i.e. resistant to benzimidazole and ivermectin, R-IVM/SA) were examined for isoenzyme variation by starch gel electrophoresis. Using stains for seven enzymes separated in five different buffer systems, no differences in the electrophoretic mobility could be detected between any of the strains. Results demonstrate a low level of enzyme variation in H. contortus and no differences in enzyme electrophoretic profile between IVM-sensitive and IVM-resistant parasites. Differences between the ivermectin-sensitive and both ivermectin-resistant strains were observed with the propionyl esterases and although some of the differences are probably associated with benzimidazole resistance, others are associated with resistance to ivermectin. The three strains of H. contortus are generally identical; however, differences between all strains of H. contortus and a strain of Dictyocaulus viviparus were detected.
Immune responses of sheep to surface antigens of infective larvae of Ostertagia circumcincta.
Immune responses to surface antigens of infective larvae of Ostertagia circumcincta were studied in 5-month old Finn-Dorset male lambs. The sheep were vaccinated subcutaneously with 2 doses of 25 micrograms/kg body weight of infective larvae surface or somatic extracts and Freund's or beryllium hydroxide adjuvants. It was found that only in lambs vaccinated with L3 surface extracts and beryllium hydroxide as an adjuvant did worm burdens differ significantly (P less than 0.01) from those of challenge controls (71.7% protection). Sheep vaccinated with the same antigenic preparation but administered with Freund's adjuvant or with beryllium adjuvant and somatic extracts demonstrated rather poor protection (32.4 and 30.5% respectively). All the vaccinated sheep showed a high IgG response to L3 surface antigens while significant levels of serum and bile IgA reacting with the surface extracts were only detected in those lambs vaccinated with L3 surface extracts and beryllium hydroxide. In immunofluorescence tests serum IgA of this group reacted with the whole surface of exsheathed larvae or with the site of opening of excretory pore while IgG antibodies reacted strongly with the anterior and posterior parts of the infective larvae. Both IgA and IgG responses to surface antigens were stage specific.
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.
The molecular epidemiology of parasites.
The explosion of new techniques, made available by the rapid advance in molecular biology, has provided a battery of novel approaches and technology which can be applied to more practical issues such as the epidemiology of parasites. In this review, we discuss the ways in which this new field of molecular epidemiology has contributed to and corroborated our existing knowledge of parasite epidemiology. Similar epidemiological questions can be asked about many different types of parasites and, using detailed examples such as the African trypanosomes and the Leishmania parasites, we discuss the techniques and the methodologies that have been or could be employed to solve many of these epidemiological problems.