Alan M. Johnson

Strain-dependent, route of challenge-dependent, murine susceptibility to toxoplasmosis

The response to an intraperitoneal (i.p.) parasite challenge was compared with that caused by an oral challenge, in five different strains of mice. The results are consistent with the hypothesis that murine susceptibility to toxoplasmosis is route of challenge-dependent as well as strain-dependent. The phenomenon occurs in both sexes and appears to be a recessive trait. The finding that the susceptibility of C57BL/6j mice to oral or i.p. challenge is almost the reverse of that of LACA mice, and that BALB/c mice are resistant to challenge by either route, offers an excellent laboratory model for studies on susceptibility to toxoplasmosis.

Phylogenetic relationships of Sarcocystis species from sheep, goats, cattle and mice based on ribosomal RNA sequences

Partial sequences of the small subunit ribosomal RNA of four species of Sarcocystis were obtained by reverse transcription of total cellular RNA. The semi-conserved regions of these four species were aligned with homologous sequences of two other Sarcocystis species and a range of other eukaryotes including Toxoplasma, Eimeria and Cryptosporidium. Parsimony analysis of the aligned sequences showed that Sarcocystis and Toxoplasma had a more recent common ancestor with Eimeria than with Cryptosporidium. The six Sarcocystis spp. did not cluster together in this analysis; two monophyletic groups were observed, one formed by the two Sarcocystis spp. with felids as definitive hosts, and another by the four Sarcocystis spp. with canids as definitive hosts. These two clades were segregated by Toxoplasma. An analysis of nucleotide divergence suggests that the difference between the two groups of Sarcocystis spp. is similar to that between invertebrates and vertebrates. The results obtained here question the validity of a separation of the genus Sarcocystis from Toxoplasma and refute classifications that place these two genera into two different subfamilies of the Sarcocystidae.

Recognition of recombinantToxoplasma gondii antigens by human sera in an ELISA

We developed an enzyme-linked immunosorbent assay (ELISA) that uses one of two recombinant polypeptides, termed H4/GST and H11/GST, as diagnostic antigens for the detection of antibodies toToxoplasma gondii in human sera. A total of 59 sera from humans with acute toxoplasmosis, 194 sera from patients with chronic toxoplasmosis, and 151 sera from subjects who were not infected withT. gondii were examined. In all, 68% of the sera from humans with acute toxoplasmosis reacted positively with one or both recombinantT. gondii antigens. By contrast, only 14% of those from patients with chronic toxoplasmosis recognized H4/GST or H11/GST. None of the sera from humans who were not infected withT. gondii, including patients with echinococcosis, entamoebosis, toxocarosis, trichinellosis, glandular fever, or rheumatoid arthritis, recognized H4/GST or H11/GST.

Phylogenetic relationships of Cryptosporidium determined by ribosomal RNA sequence comparison

Reverse transcription of total cellular RNA was used to obtain a partial sequence of the small subunit ribosomal RNA of Cryptosporidium, a protist currently placed in the phylum Apicomplexa. The semi-conserved regions were aligned with homologous sequences in a range of other eukaryotes, and the evolutionary relationships of Cryptosporidium were determined by two different methods of phylogenetic analysis. The prokaryotes Escherichia coli and Halobacterium cuti were included as outgroups. The results do not show an especially close relationship of Cryptosporidium to other members of the phylum Apicomplexa.

CLONING OF TOXOPLASMA GONDII GENE FRAGMENTS ENCODING DIAGNOSTIC ANTIGENS

Two Toxoplasma gondii gene fragments, which encode polypeptides that can be used as diagnostic antigens in an enzyme-linked immunosorbent assay were cloned and their nucleotide sequence was determined. One of the fragments (derived from the H4 gene) is 682 bp long. The mRNA of the single-copy H4 gene is 1300 nt long. The fragment derived from the other gene, termed H11, is 197 bp long. The mRNA of the single-copy H11 gene is 1900 nt long. The native polypeptides encoded by the H4 and H11 genes are 25 and 41 kDa, respectively. Based on computer analysis of the deduced amino acid sequences of the polypeptides encoded by the gene fragments, both appear to be very hydrophilic and that encoded by the H11 fragment has a high antigenic index profile. These results are consistent with the diagnostic usefulness of the polypeptides encoded by the gene fragments.

The antigenic structure of toxoplasma gondii: A review

Even though T. gondii is ubiquitous in the animal kingdom, definitive information on its antigenic structure has only become available over the last few years, largely as a result of recent advances in immunology and biochemistry. New knowledge in this area will enable the immune response to the parasite to be studied in greater detail and may lead to the development of newer, more meaningful diagnostic tests for toxoplasmosis, and possibly a vaccine against it. This paper reviews knowledge on the antigenic structure of this extremely widespread and important protozoan parasite.&NA; Even though T. gondii is ubiquitous in the animal kingdom, definitive information on its antigenic structure has only become available over the last few years, largely as a result of recent advances in immunology and biochemistry. New knowledge in this area will enable the immune response to the parasite to be studied in greater detail and may lead to the development of newer, more meaningful diagnostic tests for toxoplasmosis, and possibly a vaccine against it. This paper reviews knowledge on the antigenic structure of this extremely widespread and important protozoan parasite.

Characterization and in vitro translation of Toxoplasma gondii ribonucleic acid

RNA was extracted from purified tachyzoites of Toxoplasma gondii (RH strain) by sequential centrifugation in guanidine hydrochloride, urea, and lithium chloride. The subunits of the RNA were characterized by denaturing and non-denaturing electrophoresis in agarose gels. Poly(A)+-RNA, purified by oligo(dT)-cellulose affinity chromatography, was translated in a rabbit reticulocyte lysate assay and the products were immunoprecipitated with an experimentally infected mouse serum and a naturally infected human serum. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, fluorography of the polypeptides confirmed that the mRNA translated specific parasite antigens.

Cloning, expression and nucleotide sequence of the gene fragment encoding an antigenic portion of the nucleoside triphosphate hydrolase of Toxoplasma gondii

Toxoplasma gondii expresses high levels of an active nucleoside triphosphate hydrolase (NTPase; EC 3.6.1.3) with several unique properties. It has been detected as a circulating antigen in mice, making it an ideal candidate for diagnostic tests for toxoplasmosis. A cDNA library constructed from T. gondii poly(A)+RNA was made in lambda gt11. One hundred thousand members of this library were immunoscreened with a rabbit polyclonal antibody to the purified NTPase. Six positive clones were subcloned into the plasmid, pGEX-IN, and the inserts were restriction-mapped. All clones had identical partial restriction enzyme maps. One insert was subcloned into M13mp18 and sequencing by the deoxy/dideoxy method showed an NTPase-encoding gene (ntp) fragment of 571 bp. The insert was also purified, radiolabelled, and used to hybridize to Northern blots of tachyzoite RNA and quantitative Southern blots of tachyzoite DNA. Northern blotting revealed that the NTPase mRNA was in great abundance and had a length of about 2800 nucleotides. Southern blotting showed a gene copy number of between one and five, and the possibility that ntp is tandemly repeated over a large length of DNA. The NTPase was expressed as a glutathione S-transferase (EC 2.5.1.18) fusion protein of about 50 kDa, which reacted with polyclonal rabbit antibody on Western blotting.

The phylogenetic relationships of the genus Eimeria based on comparison of partial sequences of 18S rRNA

Reverse transcription of ribosomal RNA (rRNA) was used to obtain partial sequences of the small subunit rRNA (ssrRNA) of three species of Eimeria: Eimeria maxima, Eimeria stiedae and Eimeria tenella. These sequences were aligned with the homologous ssrRNA sequence segments of a range of other organisms in the phylum Apicomplexa, along with representatives of the ciliates, fungi, flagellates, higher plants and higher eukaryotes. Two phylogenetic tree building methods were used to investigate the phylogenetic relationships among the Eimeria species, and between the Eimeria and the other organisms represented, using Vairimorpha necatrix as an outgroup. The results show that Eimeria is almost certainly monophyletic with the apicomplexans Sarcocystis, Toxoplasma and Cryptosporidium. The three species of Eimeria differ among each other at a maximum of 9 out of 600 nucleotides at the ssrRNA locus. Such intrageneric variation is high by vertebrate standards, but low by protist standards.

Ultrastructural and biochemical studies on the immunohistochemistry of Toxoplasma gondii antigens using monoclonal antibodies.

SummaryTo determine the cellular distribution of Toxoplasma antigens, RH strain tachyzoites were incubated with either one of three monoclonal antibodies (FMC 19, FMC 20, FMC 22) to T. gondii, or one of two controls (the murine myeloma protein MOPC 21, or phosphate buffered saline), and then incubated with peroxidase-labelled goat-antimouse IgG. Diaminobenzidine was added as substrate and electron microscopy was used to localize the reaction. All three antibodies bound to the entire periphery of the tachyzoite surface membrane. To ascertain the chemical composition of the antigens against which seven monoclonal antibodies (FMC 18, FMC 19, FMC 20, FMC 22, FMC 23, 2G11, 3E6) to T. gondii reacted, untreated, pronase-treated, or periodate-treated tachyzoites were incubated with the antibodies or MOPC 21, and then with |125I|-Protein A. The pronase-treated tachyzoites showed reduced binding for six of the antibodies, compared with the reduction in binding of MOPC 21 with the pronase-treated parasites. The periodate-treated tachyzoites had reduced binding for FMC 18 only. The results of these experiments confirm that most Toxoplasma surface antigens are protein in nature, and are consistent with the hypothesis that at least one cytoplasmic antigen is secreted onto the parasite cell surface.