A method to obtain large quantities of Toxoplasma gondii tachyzoites with extreme purity

In order to extract genetic material from the intracellular parasite Toxoplasma gondii, large numbers of organisms free of host cells are necessary. A method is described in which the RH strain of T. gondii was cultivated in nonadherent U937 cells. The purification was done by countercurrent elutriation. The resulting population of 10(10) T. gondii contained only 0.17% host cell material. The method is simple, rapid, and minimizes the use of animals.     

Toxoplasma gondii: metabolism of intracellular tachyzoites is affected by host cell ATP production

PURPOSE: To evaluate different-caliber biopsy cutting needles in terms of the benefits and potential risk of bleeding in a swine model. MATERIALS AND METHODS: A total of 190 sequential liver biopsy specimens were obtained in 11 Yorkshire pigs (weight, 50-70 lb [22.5-31.5 kg]) by using 14-, 18-, and 20-gauge cutting needles. For each biopsy procedure, blood loss was determined by weighing sponges used to absorb bleeding, and sample-tissue DNA content was measured with spectrofluorometry. Analysis of variance was used to compare results. RESULTS: The larger the caliber of needle, the greater the absolute blood loss (for 14-gauge, 1.69 g; for 18-gauge, 0.74 g; for 20-gauge, 0.32 g) and DNA content per sample (for 14 gauge, 40.38 microg; for 18-gauge, 12.18 microg; for 20-gauge, 5.86 microg). The ratio of blood loss to amount of DNA recovered did not differ among the different-caliber needles. To obtain the same amount of diagnostic tissue, more passes were needed with the smaller-caliber needles. CONCLUSION: Use of larger-caliber needles is more efficient despite the greater amount of blood loss, because more tissue can be recovered and because fewer passes are necessary, which reduces the chances of complications.     

Detection of Toxoplasma gondii tachyzoites and bradyzoites in blood, urine, and brains of infected mice

Previously, a strong stimulatory response of peripheral blood mononuclear cells (PBMC) to a fermented mistletoe extract (Iscador Pini, i.p.) could be observed in normal and allergic individuals. Aim of this study was therefore to analyse the cell subtypes involved in this in vitro reactivity. PBMC from 11 non-mistletoe treated healthy and allergic individuals were therefore investigated. Flow cytometric analyses revealed that this mistletoe extract stimulated T-cells (CD3+), especially T-helper cells (CD4+), as well as monocytes at concentrations of 100 or 1,000 micrograms/ml. Furthermore, an increased proportion of T- and T-helper cells expressing the interleukin-2 (IL-2) receptor (CD25) and monocytes expressing HLA-DR molecules, respectively, could be demonstrated. There was no evidence for a major involvement of B- (CD19+), natural killer- (NK-; CD56+) and T-suppressor cells/cytotoxic T-lymphocytes (CD8+). To get more insights whether the T-cell proliferation induced by i.p. was the consequence of a primary or a secondary type of immune response, kinetic studies (n = 5) were performed hereby comparing the reactivity of the non-recall antigen keyhole limpet hemocyanin (KLH) as well as the recall antigen purified protein derivative (PPD) derived from Mycobacterium tuberculosis with that of i.p. I.p. at a concentration of 100 micrograms/ml always exhibited the kinetics of a primary immune reaction. These findings were further substantiated by additional flow cytometric studies analysing changes in the proportions of naive (CD45RA+) and memory/effector cells (CD45RO+) during the exposure to i.p. It could be shown that the proportion of naive cells decreased and that of the transition stage increased, which could indicate that there was a change towards memory/effector cells. In conclusion, these data demonstrate that i.p.-related antigens act like "non-recall"-antigens, and the in vitro immune response involves T-cells as well as monocytes.     

Stimulation of human T lymphocytes obtained from Toxoplasma gondii-seronegative persons by proteins derived from T. gondii

Toxoplasma gondii antigens are superantigens in mice. To investigate a superantigen effect in humans, lymphocytes from T. gondii-seronegative subjects were studied for proliferation to T. gondii antigens (TA). Marked cellular proliferation, predominantly of CD4+ lymphocytes, was apparent. TA elicited expansions of Vbeta-bearing lymphocytes in all subjects, but different Vbeta-bearing lymphocytes were expanded in different subjects in both CD4+ and CD8+ subpopulations. Cord blood cells also proliferated to TA. Previously fixed antigen-presenting cells were unable to present TA. Thus, T. gondii appears to produce a molecule(s) that induces polyclonal activation of human T cells and requires antigen processing to mediate this effect. That T. gondii does not appear to behave as a superantigen in humans is important in understanding the pathogenesis of T. gondii infection in immunocompromised hosts and in the design of anti-T. gondii vaccines.     

Differential study of two strains of Toxoplasma gondii

This study aimed to differentiate between the virulent and avirulent strains of T. gondii by studying morphometric measurement using C.I.A.S., determination of the relative DNA content and extract, SDS poly acrylamide gel electrophoresis and isoenzyme on cellulose acetate gel. Identifying these differences may be useful in studying different disease types and in pathogenic mechanisms. Besides, the presence of common proteins between them may be of value for diagnostic purposes and for formulation of vaccines.     

Genotypic analysis of Toxoplasma gondii isolates from pigs

To determine the prevalence of the 3 primary clonal lineages of Toxoplasma gondii (strain types I, II, and III) in a potential food source of infection for humans, we analyzed 43 isolates of T. gondii that had been collected from pigs at an abattoir in Iowa. Parasites were harvested as in vitro-grown tachyzoites, and their genotypes were determined at the SAG1 and SAG2 loci. On the basis of the allele identified at the SAG2 locus, isolates were grouped into 1 of the 3 primary lineages. Type II strains were by far the most prevalent, accounting for 83.7% of the isolates. The type III genotype was identified in only 16.3% of the isolates. These prevalences differ significantly from a previous sampling of isolates from animals but are similar to the frequencies with which they occur in human disease cases. Similar to the previously characterized strain P89, strains P62 and P105 appeared to have recombinant genotypes. The type I genotype was not identified in the isolates from pigs although these strains have previously been shown to account for approximately 10-25% of toxoplasmosis cases in humans.     

The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1

Toxoplasma gondii is an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched a Toxoplasma expressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related to SAG1. Like SAG1, these new SRS genes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2 and SRS3) are shown to be expressed on the surface of Toxoplasma tachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of the SAG1 gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acute T. gondii infections.     

Kinetics study and characterisation of target excreted/secreted antigens of immunoglobulin G, M, A and E antibodies from mice infected with different strains of Toxoplasma gondii

A kinetics study and characterisation of target excreted/secreted antigens of immunoglobulin (Ig) G, M, A and E antibodies were realised by Western blotting with immune sera of mice inoculated with three strains of Toxoplasma gondii: RH, C56 and S3. IgG antibodies of the immune sera recognised the major proteins of the three excreted/secreted antigen preparations with molecular masses of 30, 45, 63 and 77 kDa. IgM antibodies recognised proteins revealed by IgG antibodies but with variable intensities; some proteins were revealed during a short period. IgA antibodies did not recognise the 35-kDa antigen or the antigens inferior to 28 kDa. The RH excreted/secreted antigens were revealed with the highest intensity. The IgE antibodies were briefly detected in trace amounts during period from the 20th to the 35th day. The RH strain with its excreted/secreted antigens had the best antigenicity and is a good model for immunoprotection studies.     

Characterization of Toxoplasma gondii from the feces of naturally infected cats

Feces of 1,000 cats from a humane shelter in Columbus, Ohio, were examined microscopically for oocysts of Toxoplasma gondii and by inoculation into mice. From the first 541 cats examined, oocysts of Toxoplasma were found in the feces of seven cats but in none of the remaining 459 cats. Results of the dye test in these seven cats showed titers of antibody of less than 1:2 in four cats, and of 1:8, 1:6, and 1:32 in the remaining three cats. The pathogenicity and infectivity of oocysts and cysts of all seven strains were compared in mice after oral and intraperitoneal inoculations. Oocysts and cysts were more pathogenic when administered by the oral route than by the intraperitoneal route. The cysts were less pathogenic than the oocysts. Excellent cross-immunity between six of these seven feline strains and the M-7741 strain was deomonstrated in cats by the fact that oocysts were not shed in feces of cats challenged with cysts of homologous or heterologous strains.     

Nucleotide sequence of ToxPK1 gene from Toxoplasma gondii

We report here for the first time a complete nucleotide sequence (6.8 kb) of a protein kinase gene (ToxPK1) from the obligate intracellular protozoan parasite of man, Toxoplasma gondii. This gene comprising putatively of 9 exons and 8 introns forms the Toxoplasma gene with the largest number and size of introns reported so far. The predicted protein with 508 amino acids contains the 15 invariant residues as well as the characteristic motifs specific to protein serine/threonine kinases. Homology-based computational comparisons suggested that TOXPK1 belongs to or closely resembles the SNF1 subfamily of protein-serine/threonine kinases. Based on the functions of SNF1 homologs in other organisms and our RT-PCR results, it is likely that TOXPK1 may be transiently expressed to up-regulate glycogen biosynthesis during the development of tachyzoites into bradyzoites.     

Serologic survey for Toxoplasma gondii in grizzly bears from Alaska

Aquaporin-4 is a mammalian water channel protein that is predominately expressed in brain, where it is believed to mediate water homeostasis. Here we report the isolation and characterization of the cDNA for mouse Aqp4 and map the gene to the proximal region of mouse chromosome 18. This region contains the neurological mutation ataxia, but further analysis reveals that Aqp4 is not responsible for the ataxia phenotype.     

Consensus sequence of translational initiation sites from Toxoplasma gondii genes

Clinical analysis and the set of laboratory studies, performed in 25 patients one, two, three or six years after surviving acute period of trichinellosis, documented complaints in 22 patients (88.0%) in the form of muscle complaints (68.2%), cardiovascular complaints (45.4%), generalized weakness (40.9%) and fatigability (31.8%). No significant alterations were demonstrated in electrocardiographic records. In 71.4% examined patients lactic dehydrogenase activity was augmented. Presence of IgG antibodies against the E/S antigen of Trichinella sp. was disclosed in 24 (96%) patients, including 22 patients (88.0%), in whom high titres of the antibodies were found. Morphological studies on muscle tissue (performed in 5 patients) disclosed alterations typical of trichinellosis in 4 patients and presence of Trichinella larvae, calcified to a significant extent, in 2 patients. The long term persistence of IgG class antibodies against Trichinella antigen in patients who survived acute period of trichinellosis a few years earlier points to a chronic antigenic stimulation, probably reflecting progressive destruction of Trichinella larvae in muscle tissue. This may also be expressed in complaints reported by the patients. The problem requires further observations and clinical studies.     

Toxoplasma gondii: an ultrastructural study of host-cell invasion by the bradyzoite stage

The invasion of Toxoplasma gondii tachyzoites and bradyzoites was followed in bovine kidney cells via electron microscopy. The process of invasion differed between bradyzoites and tachyzoites. In the early stages of entry there was evidence of localised formation of membrane projections in the host cell adjacent to the parasite. Parasite reorientation and rhoptry release appeared to be necessary for invasion; however, the tight junction could not be clearly discerned and there was no evidence of constriction or of any membrane shedding from the parasite. The resulting parasitophorous vacuole was smaller than the tachyzoite vacuole and parasites were frequently found to lie immediately under the host cell membrane. The vacuole was rapidly adapted by the release and formation of an intra-phagosomal membrane network, while the parasitophorous vacuole formed a relationship with host-cell endoplasmic reticulum.     

A cell culture system for study of the development of Toxoplasma gondii bradyzoites

Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS-induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite-specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno-electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst-like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free-floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of gamma-interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL-6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.     

Infection and stage conversion during murine pulmonary toxoplasmosis: a study with three different strains of Toxoplasma gondii

OBJECTIVE: To investigate whether intravenously administered liposomal alpha-tocopherol can protect the lung from the injurious action of Escherichia coli lipopolysaccharide (LPS). DESIGN: Prospective, randomized animal study. SETTING: Government research laboratory. SUBJECTS: Twenty adult male Sprague-Dawley rats. INTERVENTIONS: Animals were intravenously pretreated with alpha-tocopherol liposomes (20 mg alpha-tocopherol/kg body weight), plain liposomes, or saline. Twenty-four hours later, pretreated animals were challenged with an intravenous injection of LPS (E. coli 0111:B4, 1 mg/kg body weight), and killed 2 hrs after LPS challenge. MEASUREMENTS AND MAIN RESULTS: Challenge of saline-pretreated animals with LPS resulted in lung injuries as evidenced by an increase in wet lung weight and a reduction in pulmonary angiotensin converting enzyme (25%) and alkaline phosphatase (28%), injury markers of lung endothelial and epithelial type II cells, respectively. Also, LPS administration resulted in an increase in pulmonary myeloperoxidase and protease activities, indicative of a neutrophilic inflammatory response. Pretreatment of animals with liposomal alpha-tocopherol significantly attenuated the LPS-induced edematous lung weight response, and reduced the extent of injuries to the pulmonary endothelial and epithelial cells, demonstrated by a significantly smaller reduction in the corresponding enzyme marker activities. CONCLUSION: These results suggest that augmentation of the pulmonary antioxidant status can ameliorate LPS-induced lung injuries mediated by oxidative stress mechanisms.