Heat shock proteins (HSPs) area group of proteins involved in maintaining cell homeostasis. Their synthesis is increased dramatically in response to stimuli such as temperature shifts, changes in concentrations of glucose and calcium and also in response to immune factors. HSP70 has been found to be strongly expressed in virulent strains of Toxoplasma gondii taken from immunocompetent mice but poorly expressed in avirulent strains taken from immunocompromised mice or virulent and avirulent strains cultivated invitro. Hence, T. gondii HSP70 may help protect parasites against the host’s immune response, allowing the virulent strains to persist as tachyzoites without the enforced encystation found in avirulent strains. This makes T. gondii HSP70 an attractive subunit candidate. To investigate the immunogenicity of this protein it was affinity purified from tachyzoites of the virulent RH strain and injected into BALB/c mice along with Freund’s Complete Adjuvant (FCA). A booster shot with Freund's Incomplete Adjuvant (FIA) as given 4 weeks after the initial injection and blood was collected at 8 weeks. The RH strain HSP70 DNA was cloned as a GST fusion protein, expressed and also injected into BALB/c mice to see if the immune response generated by the recombinant protein matched that of the native protein. Antibody response was measured by ELISAs to detect IgG1, IgG2a, IgG2b and IgG3. An elevation of IgG1 antibodies, but not other IgG subsets, was observed in the mice immunised with native or recombinant HSP70. This suggests that the immune response seen with the recombinant protein is similar to that seen with the native protein. However, the response to either protein was weak, suggesting that the T. gondii HSP70 may not be a particularly good immunogen.
The plastid DNA from different strains of Toxoplasma gondii has been isolated using a variety of techniques. The relative effectiveness of these isolation techniques is compared. Initially, clones were isolated from genomic DNA libraries, but 100% positive identification of these clones proved difficult, due to variations in the plastid sequences and the lack of comparable sequences. Purification techniques using commercial columns gave eluates highly enriched in plastid DNA, but other contaminating DNA was also shown to be present. Finally, the novel long polymerase chain reaction technique (long-PCR) was trialed and was found to be the most efficient isolation and purification method.
The nucleoside triphosphate hydrolase (NTPase) of Toxoplasma gondii demonstrates an unusually high level of ATP hydrolysis, which, among the apicomplexan parasites, has only been observed in T. gondii and the closely related Neospora caninum. In T. gondii, NTPase has been shown to be highly expressed (constituting up to 8% of the tachyzoite protein) and is an immunodominant antigen in mice and humans. Two isoforms exist – NTPaseⅠ and NTPaseⅡ. NTPaseⅠ demonstrates a 4.5 fold greater activity than NTPaseⅡ with respect to ATP hydrolysis. Past studies suggest that only virulent strains possess the highly active NTPaseⅠ isoform. We have recently identified a B cell epitope (aa 484-502) on the NTPase isoforms which, despite some cross reactivity, is differentially recognized by a naturally infected human serum sample. In this study we used competitive antigen ELISAs and have identified that this serum sample reacts specifically to the NTPaseⅠ epitope, whilst the corresponding region on NTPaseⅡ isoform is the less specific cross reactive epitope. These results are consistent with the hypothesis that this patient has been infected with a virulent strain of T. gondii.
The host parasite relationship of the enteric (coccidian) and exoenteric (tachyzoite, bradyzoite) forms of Toxoplasma gondii was examined in vivo by electron microscopy and immunocytochemistry. Significant structural and molecular differences were observed between the enteric and exoenteric forms. The expression and distribution of certain rhoptry and all the dense granule proteins was examined by immunocytochemistry. It is was observed that, while the antibody recognizing ROP2,3,4 positively stain the anterior of the infective stages (merozoite, tachyzoite and bradyzoite) of both the enteric and exoenteric stages, ROP1 appeared to be absent from the merozoite. It was found that GRA8, like GRAs1-6, was present in both the tachyzoite and bradyzoite stages but did not appear to be expressed by the enteric stages. The only dense granule proteins expressed by the enteric (coccidian) stages were GRA7 and NTPase. By electron microscopy, the parasitophorous vacuole (PV) containing the enteric forms in the enterocytes of the cat was limited by a laminated wall consisting of three fused unit membranes. This is in contrast to the exoenteric stages where the PVs are limited by a single unit membrane. These differences point to a unique host/parasite relationship for the enteric stages of T. gondii.
Babesia equi merozoite antigen-2 (EMA-2) expressed by recombinant baculovirus in insect cells was coupled latex beads, and these complexes were used to develop the latex agglutination test for detection of antibodies to B. equi. Serum samples from horses experimentally infected with either B. equi or Babesia caballi and from normal horses were assayed by the test. When the latex agglutination test compared with the enzyme-linked immunosorbent assay, the results of both tests agreed completely, suggesting that the latex agglutination test is suitable for a routine test.