NO FULL TEXT AVAILABLE. Access is restricted indefinitely. ----- Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect
most, if not all, warm blooded vertebrates. The effects of T. gondii infection have been
widely studied in the murine host, which is highly susceptible to infection and where
isolates have shown a remarkable dichotomy in virulence, allowing strains to be classified
as virulent or avirulent based on the number of tachyzoites required to cause severe disease.
In humans, T. gondii causes a wide range of clinical manifestations that are dependent on
factors that are intrinsic to both the host and the parasite. Most infections are
asymptomatic in the immunocompetent host, however, a small number can suffer from
symptomatic ocular infection. Severe symptomatic illness is most common in
immunocompromised hosts and can cause serious disease leading to death if left untreated.
For instance, congenital toxoplasmosis, which can occur if the mother is infected for the
first time during pregnancy, may result in spontaneous miscarriage, stillbirths or foetal
abnormalities. Patients who are immunocompromised by HIV infection or who are organ
donor recipients can experience a recrudescence of latent T. gondii infection often leading
to fatal encephalitis.
A number of studies have attempted to elucidate if the infecting strain influences the
clinical disease outcome. These studies have relied on isolation of the parasite from
symptomatic patients followed by either a murine virulence test or strain genotyping.
Given that symptomatic patients may only comprise about 1 % of infected individuals, data
on the infecting strain type in the larger infected population is needed to give a complete
picture of the epidemiology of T. gondii strains in human infection that is not subject to
sampling and parasite isolation biases.
The aim of this project was to determine whether a serological based assay could be
developed for identifying the infecting strain type in human toxoplasmosis. In this project
the nucleoside triphosphate hydrolase (NTPase) of T. gondii was evaluated as a potential
serological marker of the virulence of the infecting strain.
The NTPase of T. 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. This highly active NTPase was chosen for this study
because it is highly expressed (constituting up to 8% of the tachyzoite protein) and is an
immunodominant antigen in mice and humans. Two isoforms (NTPaseI and NTPaseII),
which correlate to parasite virulence and exhibit different activities with respect to
hydrolysis of ATP, exist. Previous studies suggested that all strains of T. gondii contained
the less active NTPaseII whilst only virulent strains contained the more active NTPaseI
In order to further investigate the correlation between NTPase isoform and
biological significance, the full length NTPaseI and NTPaseII isoforms were cloned and
expressed as glutathione S-transferase fusion proteins in Escherichia coli. Enzyme linked
immunosorbant assays (ELISAs) were then used, with the full length recombinant NTPases
as antigens, to examine 188 naturally infected T. gondii positive sera and 83 T. gondii
negative sera for antibody reactivity. All positive sera reacted to T. gondii whole tachyzoite
lysate antigen, 31 sera reacted to both NTPase isoforms, three sera reacted specifically to
NTPaseI and two sera specifically to NTPaseII. Immunoblot analysis of the five sera
reacting to either NTPase I or NTPaseII revealed both quantitative and qualitative
differences in reactivity to the two isoforms. One of the five serum samples reacted only
with NTPaseI and did not show any reactivity to NTPaseII. When this serum sample was
used in comparative immunoblot analysis using truncations of the NTPase I isoform, a
presumptive differential epitope between the NTPaseI and NTPaseII isoforms was
identified within an 81 amino acid region (amino acids 445 - 526) at the C-terminus of the
NTPaseI isoform. This differential reactivity was further localised to the 12 residue region
of greatest variability between the two isoforms (residues 488 - 499) using synthetic
peptides. This is the first report of naturally infected human sera being used to identify a
Competitive antigen ELISAs using the synthetic peptides revealed that this serum
sample reacted specifically to the NTPaseI epitope, whilst the corresponding region on
NTPaseII isoform was a less specific cross-reactive epitope. These results were consistent
with the hypothesis that this patient had been infected with a virulent strain of T. gondii.
Further evidence of the differential reactivity of these peptides was observed when sera
from rats that were immunised with either recombinant NTPaseI or NTPaseII reacted
specifically and differentially to the peptides in ELISAs.
To determine if it was possible to identify the infective strain type (virulent or
avirulent) in T. gondii infections by serological means, the isoform-specific peptides from
the NTPaseI and NTPaseII were used in ELISAs with sera from rats and humans where the
infecting strain was known. The ELISAs were used to test six groups of rats that were
infected with one of three virulent (RH, P strain or Ent) or three avirulent (Me49, C strain
or TPR) strains of T. gondii. No differential antibody reactivity was detected by either
whole recNTPase ELISA or peptide ELISA in the sera of rats infected by either virulent or
avirulent strains of T. gondii.
A panel of human sera from patients infected with known laboratory strains of
T.gondii or naturally infected patients where the parasite was isolated and its virulence
determined in mice was also studied. Differential reactivity to whole recNTPase isoforms
was detected in some human sera but this reactivity was not detected by the isoform
specific peptide ELISAs.
Overall, although the NTPase peptides exhibited differential antibody reactivity,
this was not correlated to the virulence status of the infecting strain and they cannot,
therefore, be used as serological markers of virulence of the infecting strain.