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PubMed2003

Molecular and bioassay-based detection of Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis).

Toxoplasma gondii is associated with morbidity and mortality in a variety of marine mammals, including fatal meningoencephalitis in the southern sea otter (Enhydra lutris nereis). The source(s) of T. gondii infection and routes of transmission in the marine environment are unknown. We hypothesise that filter-feeding marine bivalve shellfish serve as paratenic hosts by assimilation and concentration of infective T. gondii oocysts and their subsequent predation by southern sea otters is a source of infection for these animals. We developed a TaqMan PCR assay for detection of T. gondii ssrRNA and evaluated its usefulness for the detection of T. gondii in experimentally exposed mussels (Mytilus galloprovincialis) under laboratory conditions. Toxoplasma gondii-specific ssrRNA was detected in mussels as long as 21 days post-exposure to T. gondii oocysts. Parasite ssrRNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Parasite infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill, or digestive gland), but only digestive gland samples remained bioassay-positive for at least 3 days post-exposure. For each time point, the total proportion of mice inoculated with each of the different tissues from T. gondii-exposed mussels was similar to the proportion of exposed mussels from the same treatment groups that were positive via TaqMan PCR. The TaqMan PCR assay described here is now being tested in field sampling of free-living invertebrate prey species from high-risk coastal locations where T. gondii infections are prevalent in southern sea otters.

Animals↗
PubMed2003

Translocation of ribosomal protein P0 onto the Toxoplasma gondii tachyzoite surface.

A ribosomal phosphoprotein P0 detected on the surface of the human malarial parasite Plasmodium falciparum (PfP0) has been shown to be recognised by invasion blocking antibodies. Using cross-reactive polyclonal antibodies against PfP0, the surface localisation has also been demonstrated on certain mammalian cells, yeast and Toxoplasma gondii. We sought to characterise the phenomenon of surface localisation in Toxoplasma using T. gondii P0 protein. Sequence analysis of a cDNA clone isolated from a T. gondii library showed marked similarity to PfP0, suggesting that T. gondii expresses an orthologous gene, TgP0. The expression of TgP0 was corroborated by Northern blot analysis revealing a transcript of 1.8 kb in size. Immunofluorescence analysis using anti-PfP0 indicated surface localisation of TgP0. To confirm surface translocation of the TgP0, tachyzoites were transfected with the HA-tagged TgP0 gene followed by immunofluorescence detection of the HA-tag. Surface translocation of transiently expressed TgP0 and blocking of tachyzoite invasion of human foreskin fibroblasts by anti-PfP0 antibodies suggest that P0 protein plays an important role in T. gondii invasion of human cells.

Animals↗
PubMed1998

Effect of gamma irradiation on unsporulated and sporulated Toxoplasma gondii oocysts.

The effect of 137Cs irradiation on unsporulated and sporulated Toxoplasma gondii oocysts was investigated as a model system for sterilisation of fruit contaminated with other coccidia such as Cyclospora or Cryptosporidium. Unsporulated oocysts irradiated at > or = 0.4 to 0.8 kGy sporulated but were not infective to mice. Sporulated oocysts irradiated at > or = 0.4 kGy were able to excyst, and sporozoites were infective but not capable of inducing a viable infection in mice. Toxoplasma gondii was detected in histologic sections of mice up to 5 days but not at 7 days after feeding oocysts irradiated at 0.5 kGy. Transmission electron microscopy revealed that sporozoites from irradiated oocysts penetrated enterocytes and all cells in the lamina propria except for red blood cells. Sporozoites appeared normal ultrastructurally and formed a typical parasitophorous vacuole containing a well-developed tubulovesicular membrane network. Raspberries inoculated with sporulated T. gondii oocysts were rendered innocuous after irradiation at 0.4 kGy. Results indicate that irradiation at 0.5 kGy is effective in "killing" coccidian oocysts on fruits and vegetables.

Animals↗
PubMed1998

Western blot analysis of the IgG responses of ruminants infected with Neospora caninum and with Toxoplasma gondii.

The IgG antibody responses of sheep, goats and cattle inoculated subcutaneously with live Neospora caninum tachyzoites of the NC1 isolate were analysed by Western blotting. Antibodies were detected against a wide range of NC1 tachyzoite antigens (6.5 to 80 kDa). The dominant antibody responses were directed against proteins at 36.5-38, 45.5-48.5, 52-53.5, 58, 58.5, 59.5, 60.5, 62, 63.5, 64, 66.5, 67, 67.5, 68.5 and 69.5 kDa, with sera from all three species. These sera were also used to probe blots of Toxoplasma gondii antigen and, while a number of protein bands were recognized, there was no consistency within or between animal species. The IgG antibody responses of sheep, goats and cattle orally infected with T. gondii oocysts of the M3 isolate were analysed by the same methods. Antibodies were detected to a range of S48 toxoplasma tachyzoite antigens (11 to 83 kDa). The dominant antibody responses were directed against proteins at 11, 16-17, 21.5, 22.5-23.5, 26-28.5, 32-35, 49.5, 50.5, 53, 54.5, 60.5 and 61 kDa, with sera from all three species. These sera were also used to probe blots of N. caninum antigen; antibody responses to numerous antigens were detected but showed little consistency within or between animal species.

Animals↗
PubMed1997

Particle counting assay for anti-toxoplasma IgG antibodies. Comparison with four automated commercial enzyme-linked immunoassays.

An assay for anti-toxoplasma IgG antibodies based on agglutination of latex particles was set up and compared with commercial immunoassays. The reaction was measured by instrumental counting of particles remaining unagglutinated. The running time was 45 min. This test (PaC) was compared using 243 serum samples with four automated commercial immunoassays: the Enzymum test Toxo IgG (ES300, Boehringer), the Vidas Toxo IgG (Biomérieux), the IMX Toxo IgG (Abbott), the Magia Toxoplasma gondii IgG (Merck). The mean values (+/- SD) obtained by IMX (25 IU +/- 68) and ES300 (45 IU +/- 142) were significantly lower than the values obtained by Vidas (73 IU +/- 237, p < 10(-4) and p = 0.006, respectively), by Magia (80 IU +/- 300, p < 10(-4) and p = 0.0005) and by PaC (70 IU +/- 260, p < 10(-4) and p = 0.0126). The correlations between PaC and Toxo IgG Boehringer, Biomérieux, Abbott, Merck were r = 0.97, r = 0.98, r = 0.94, r = 0.98, respectively. The correlation coefficients between the enzyme-immunoassays ranged from 0.96 to 0.99. All positive samples by PaC were found to be positive by enzyme-immunoassays except for eight sera which were doubtful positives by the Enzymum test ToxoIgG from Boehringer. No negative sample by PaC was found positive by any of the enzyme-immunoassays. In PaC, when two latex preparations coated with different antigen were compared, the correlation was rather weak (r = 0.93) suggesting that the selection of the antigen can be critical. In conclusion, the four automated commercial immunoassays now available gave similar results. However, the discrepancies observed in this study underlined the importance of clinical and biological follow-up of the patients and the necessity to confirm the result. The introduction of a new technique such as PaC, which is now available for a large variety of assays in Clinical Chemistry and Microbiology, is justified by its intrinsic advantage of homogeneity. Therefore, automation is easy as well as the control of possible interference.

Animals↗
PubMed1996

Impact of prophylactic ganciclovir on bronchoalveolar lavage lymphocyte numbers and phenotypes in murine cytomegalovirus-induced reactivation of Toxoplasma pneumonia.

In a mouse model of cytomegalovirus (CMV)-induced immunosuppression, murine CMV (MCMV) infection results in reactivation of Toxoplasma pneumonia, and prophylactic but not delayed administration of ganciclovir attenuates the severity of this pneumonia. We now report that the protection observed with ganciclovir is associated with blunting of the alterations in bronchoalveolar lavage (BAL) lymphocyte numbers and phenotypes observed in untreated mice. Specifically, prophylactic ganciclovir prevents the usual drop in BAL CD4+ lymphocytes observed in the first week after MCMV injection--that is, the period of MCMV-associated immunosuppression. Furthermore, prophylactic ganciclovir markedly blunts the usual rise in BAL T lymphocytes (CD8+ > CD4+) seen later during the peak of reactivated Toxoplasma pneumonia. These findings suggest that prophylactic ganciclovir can protect animals from virus-associated opportunistic pneumonia by attenuating virus-induced changes in BAL lymphocytes and the attendant suppression of lung immunity.

Animals↗
PubMed1999

How do Jains get toxoplasma infection?

Toxoplasma infection is as prevalent among Jains as among other religious groups in the same area. Jain laws mean that exposure to Toxoplasma gondii via well known routes of infection is unlikely; drinking water may be the vehicle of infection.

Animals↗
PubMed1990

Toxoplasma gondii retinochoroiditis and optic neuritis in acquired immune deficiency syndrome. Report of a case.

A 29-year-old man with acquired immune deficiency syndrome (AIDS) was found to have a retinochoroiditis and optic neuritis of his left eye. Results of fundus examination showed inflammatory and hemorrhagic retinal lesions consistent with retinitis due to cytomegalovirus infection. A computed tomographic (CT) scan demonstrated enlargement of the optic nerve. The eye was enucleated and results of histopathologic examination showed retinal necrosis with underlying choroiditis and an optic neuritis. Numerous encysted Toxoplasma gondii organisms were present in the retina and tachyzoites were present in the optic nerve. To the authors' knowledge this is the second histologically documented case of Toxoplasma optic neuritis in a patient with AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Feline ocular and cerebrospinal fluid Toxoplasma gondii-specific humoral immune responses following specific and nonspecific immune stimulation.

Toxoplasma gondii-naive cats and cats previously infected orally with T. gondii tissue cysts were inoculated with soluble tachyzoite antigens plus adjuvant or adjuvant alone. Toxoplasma gondii-specific IgM and IgG were measured in serum, aqueous humor, and cerebrospinal fluid (CSF). The Goldman-Witmer coefficient (C value) for ocular or central nervous system (CNS) antibody production was calculated for aqueous humor or CSF samples positive for T. gondii-specific antibodies. Following inoculation with adjuvant plus soluble tachyzoite antigens, ocular and CNS T. gondii-specific IgG C values increased in the three previously infected cats. Following inoculation with adjuvant, the two previously infected cats had increases in ocular and CNS T. gondii-specific IgG C values. Ocular (2/3 cats) or CNS (1/3 cats) T. gondii-specific IgG C values of over 1 were detected in some T. gondii-naive cats following inoculation with adjuvant plus soluble tachyzoite antigens. The results of this study suggest that T. gondii-specific IgG C values of over 1 in aqueous humor or CSF do not prove active ocular or CNS infection in all cats.

Adjuvants, Immunologic↗
PubMed1998

Interferon-gamma antagonizes transforming growth factor-beta2-mediated immunosuppression in murine Toxoplasma encephalitis.

The in vivo modulating activity of recombinant transforming growth factor (TGF)-beta2 on acute toxoplasmosis was evaluated in both Toxoplasma gondii susceptible C57BL/6 and resistant BALB/c mice. TGF-beta2 lethally exacerbated Toxoplasma encephalitis in C57BL/6, but not in BALB/c mice. In C57BL/6 mice, TGF-beta2 induced a profound dose-dependent increase of the intracerebral parasitic load as well as a reduction of IFN-gamma levels in serum and cerebrospinal fluid with a coincident decrease of MHC class II antigen expression of macrophages, microglial cells, and B cells. Furthermore, TGF-beta2-treated C57BL/6 mice showed a reduced activation of CD4+ and CD8+ T cells and a diminished recruitment of immune cells to the brain. The TGF-beta2-mediated development of lethal toxoplasmosis in C57BL/6 mice was abolished by treatment with recombinant interferon (IFN)-gamma.

Animals↗
PubMed2000

The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels.

A monoclonal antibody (MAb) has been generated against a novel 63 kDa surface/apical antigen of Toxoplasma gondii tachyzoites which is identified here as TgAMA-1, the Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1). Sequence analysis, phase partitioning in Triton X-114, and labeling of TgAMA-1 with iodonaphthalene azide all suggest that TgAMA-1 is a type I transmembrane protein. There is a high degree of sequence similarity between TgAMA-1 and Plasmodium AMA-1, most notably in the position of conserved cysteine residues within the protein's predicted extracellular domain. In contrast to full length Plasmodium AMA-1, which has previously been localized to the rhoptries, it is shown here by immunofluorescence and immunoelectron microscopy that intracellular TgAMA-1 is found in the micronemes. A 53 kDa N-terminal proteolytic fragment of TgAMA-1 is constitutively secreted from the parasite at 37 degrees C. As is the case with other microneme proteins, the proteolytic processing and secretion of TgAMA-1 is dramatically enhanced in response to treatments which increase intracellular calcium levels.

Amino Acid Sequence↗
PubMed2000

Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii.

During invasion of host cells, Toxoplasma gondii discharges the contents of small, apically located secretory organelles called micronemes. Micronemal proteins are known to be necessary for both parasite motility and invasion of host cells. To further define the contents of Toxoplasma micronemes, we used cell fractionation and secretion-modulating drugs to identify six novel, putative micronemal proteins. In this paper we describe preliminary characterization of one of these novel proteins, TgMIC5. Molecular cloning and DNA sequence analysis of the TgMIC5 cDNA and gene revealed that it encodes a previously identified immunodominant antigen called H4. TgMIC5 also possesses a consensus sequence unique to members of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases). TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by signal peptidase before being further processed to a mature protein of 22 kDa. Using a combination of protein secretion experiments, immunofluorescence and immunoelectron microscopy, we demonstrated that TgMIC2 is stored in the micronemes of T. gondii tachyzoites before it is secreted into the surrounding medium. Based on its homology with parvulin-like PPIases, TgMIC5 may assist in the folding of other micronemal proteins that function in invasion of host cells by T. gondii tachyzoites.

Amino Acid Sequence↗
PubMed2001

Characterization of the subpellicular network, a filamentous membrane skeletal component in the parasite Toxoplasma gondii.

Electron microscopic examination of detergent-extracted Toxoplasma tachyzoites reveals the presence of a mechanically stable cytoskeletal structure associated with the pellicle of this parasite. This structure, composed of interwoven 8-10 nm filaments, is associated with the cytoplasmic face of the pellicle and surrounds the microtubule-based cytoskeleton. Two protein components of this network, TgIMC1 and TgIMC2, were identified. Both are novel proteins, but have a resemblance to mammalian filament proteins in that they are predicted to have extended, coiled-coil domains. TgIMC1 is also homologous to articulins, the major components of the membrane skeleton of algae and free-living protists. A homologue of TgIMC1 in the related malaria parasite Plasmodium falciparum was also identified suggesting the presence of structurally similar membrane skeletons in all apicomplexan parasites. We suggest that the subpellicular network, formed by TgIMC1 and 2 in Toxoplasma gondii and related parasites, plays a role in the determination of cell shape and is a source of mechanical strength.

Amino Acid Sequence↗
PubMed2002

Characterization of TgROP9 (p36), a novel rhoptry protein of Toxoplasma gondii tachyzoites identified by T cell clone.

T cell clone 3Tx19 detects a Toxoplasma gondii tachyzoite protein which, in high resolution 2D gel electrophoresis, runs at 36 kDa apparent MW with two spots of pI 5.9 and 6.5, thus exhibiting a migration pattern distinct from those of other known Toxoplasma antigens. The sequences of peptide fragments from tryptic digestion of the more prominent protein spot allowed the design of oligonucleotide primers to obtain the coding cDNA sequence. Sequence analysis of cDNA from strain BK revealed a 363 amino acid open reading frame, defined by all nine peptide sequences determined. The deduced protein sequence contains two hydrophobic segments, one near the N-terminus including a predicted signal peptide and a shorter second at the carboxy terminus, but homology to any other known protein is lacking. With synthetic peptides covering the complete primary structure, the epitope for clone 3Tx19 was mapped within the deduced partial sequence, which had remained unconfirmed by tryptic peptides. Antibodies raised against another, putative B cell epitope peptide detected the same two protein spots in 2D gel, indicating that they are antigenically related isoforms. The protein p36 is expressed by T. gondii isolates of all three intraspecies subgroups, but not in the bradyzoite stage. In intracellular tachyzoites, p36 colocalizes with rhoptry proteins and has a distribution pattern disparate from that of dense granule and microneme proteins. Subcellular fractionation indicated that p36 is a soluble constituent of tachyzoites. We suggest that this T cell-stimulatory novel rhoptry protein of T. gondii be named ROP9. It represents a marker of the tachyzoite stage.

Amino Acid Sequence↗
PubMed1998

GRA7, an excretory 29 kDa Toxoplasma gondii dense granule antigen released by infected host cells.

Monoclonal antibody (mAb) TxE2, reactive with Toxoplasma gondii excretory products, detects an acidic 29 kDa protein (p29) which, in 2D gel electrophoresis, exhibits a migration pattern distinct from those of the toxoplasmic excretory proteins described so far. The sequence of seven peptides from tryptic digestion of isolated p29 allowed the design of primers to obtain the coding DNA sequence. The full-length gene was amplified from genomic DNA of T. gondii strain BK and the sequence was identical with that of the corresponding cDNA, providing evidence for an intron-free gene structure. A single mRNA transcript of 1.3 kb was detected by Northern blot analysis. The deduced 236 amino acid protein contains a putative N-terminal signal peptide, one site of potential N-linked glycosylation, and, close to the C-terminus, a further hydrophobic, putative transmembrane domain. With synthetic peptides spanning the sequence of p29, the epitope for mAb TxE2 was mapped adjacent to the putative signal sequence. The antigen, which represents almost 0.5% of T. gondii protein, is expressed in strains of all three intraspecies subgroups, and is associated with the parasite dense granules as demonstrated by immunoelectron microscopy. In tachyzoite-infected cells, p29 accumulates within the parasitophorous vacuole and co-localizes with its delimiting membrane. In bradyzoite-infected cells, p29 is present within the host cell cytoplasm as detected by immunofluorescence staining, and, furthermore, in the supernatant of cyst-bearing cell culture lacking extracellular parasites as shown by enzyme-linked immunosorbent assay (ELISA). Thus, p29 which is named dense granule protein (GRA)7 may indicate the presence of intracellular toxoplasma.

Amino Acid Sequence↗
PubMed1998

Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii.

We have engineered a mutant version of the green fluorescent protein GFP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1996;173:33-38) for expression in the protozoan parasite Toxoplasma gondii. Although intact GFP was not expressed at any detectable level, GFP fusion proteins could be detected by fluorescence microscopy, flow cytometry (FACS), and immunoblotting. Both extracellular tachyzoites and T. gondii-infected host cells could readily be sorted by FACS, which should facilitate a variety of selection strategies. Several selectable markers were tested for their ability to produce stable green transgenic parasites. Fluorescence intensity was directly correlated with gene copy number and protein expression level. Weak selectable markers such as chloramphenicol acetyl transferase (CAT) driven by the SAG1 promoter, which yield multicopy insertions, are therefore most effective for selecting green fluorescent parasites-particularly when coupled to constructs which employ a strong promoter to drive GFP expression. Transformation vectors developed in the course of this work should be of general utility for the overexpression of heterologous transgenes in Toxoplasma. CAT-GFP fusion proteins were expressed in the parasite cytoplasm. GFP fusions to the P30 major surface antigen (linked on the same plasmid to a CAT selectable marker under control of various promoters) could be detected in dense granules within living cells, and were efficiently secreted into the parasitophorous vacuole. GFP fusions to the rhoptry protein ROP1 were targeted to rhoptries (specialized secretory organelles at the apical end of the parasite).

Animals↗
PubMed1999

Isolation and characterization of a subtractive library enriched for developmentally regulated transcripts expressed during encystation of Toxoplasma gondii.

To survive within infected hosts, Toxoplasma gondii undergoes profound metabolic and morphological changes by differentiating into a cyst characterized by its resistance to the immune system and chemotherapy. The stimulus that triggers Toxoplasma encystation and the molecular mechanisms regulating the bradyzoite phenotype are still unknown. Here, we developed a differentiation method in conjunction with a selective and subtracted cDNA strategy devised to identify developmentally regulated transcripts. We isolated and analyzed 65 cDNA clones. In addition to bradyzoite specific cDNAs previously reported, we demonstrate that twelve genes are exclusively or preferentially transcribed in the encysted bradyzoite forms of T. gondii using semi-quantitative RT-PCR. Among cDNAs identified, are those encoding predicted homologues of chaperones (mitochondrial heat shock protein 60, T-complex protein 1), DNA-damage repair protein, phosphatidylinositol synthase, glucose-6-phosphate isomerase and enolase. The identification of these genes opens the way for further study of molecular mechanisms controlling gene expression during T. gondii encystation.

Animals↗
PubMed1998

Seroprevalence of Toxoplasma gondii and Trichinella spiralis in finishing swine raised in different production systems in North Carolina, USA.

We compared the seroprevalence of both Toxoplasma gondii and Trichinella spiralis in finishing pigs raised in different production systems in North Carolina, USA. Farms were either finishing sites using all-in/all-out management of buildings in multiple-site systems (14 farms) or farrow-to-finish systems using continuous-flow management of finishing barns or outdoor accommodation 14 farms). The two groups of herds differed with respect to several management variables. A total of 13 of 2238 samples (0.58%) were positive for antibodies to Toxoplasma gondii using the modified agglutination test. Of these, 12 were from 63 pigs sampled on a farm where finishing pigs were kept on pasture. Only one of 1752 (0.057%) samples from pigs kept in total confinement systems was seropositive. Only one pig of 2183 (0.046%) tested positive by ELISA for antibodies against T. spiralis. In this region, management practices in modern production systems appear to be adequate to virtually eliminate the risk of infection of finishing pigs with both T. gondii and T. spiralis.

Animal Husbandry↗