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PubMed1984

Epizootics of ovine abortion due to Toxoplasma gondii in north central United States.

Toxoplasma-induced abortions were diagnosed in 14 flocks of sheep, 4 in Minnesota, 2 in Iowa, and 8 in South Dakota, during the 1982-1983 lambing season. Toxoplasma gondii was seen in histologic sections from 6 placentas and from brains of 2 lambs. Toxoplasma antibody was found by dye test and agglutination test in thoracic fluid of 15 fetal lambs. Lesions typical of toxoplasmosis were seen in fetal placentas from 26 ewes.

Abortion, Septic↗
PubMed1983

Temperature-sensitive mutants of Toxoplasma gondii: pathogenicity and persistence in mice.

Certain temperature-sensitive mutants of the RH strain of Toxoplasma gondii and a "back mutant," all maintained in human fibroblasts, were studied in mice. Most mutants gave rise to acute, fatal infections, and after sulfonamide prophylaxis rarely persisted as chronic infections in mice. However, the ts-4 strain was nonfatal in doses up to 10(3) to 10(5) tachyzoites, elicited high titers of antibody, and did not persist beyond 2 mo. No Toxoplasma cysts were found. There was no evidence that a febrile reaction of the mice was restrictive, because the highest temperatures, 37.9 to 38.4 C, occurred 3 days after infection, whereas the organisms were recoverable for 16 to 32 days. Because doses of Toxoplasma, survived by 11 of 12 normal BALB/c mice, and by one of five thymic transplanted athymic mice, killed six of six athymic mice, it appears that the limited persistence of ts-4 is related to the immunologic response.

Animals↗
PubMed1983

Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies.

Western Blot analysis revealed that both IgM and IgG antibodies present in the sera of humans infected with Toxoplasma gondii recognize three major antigens with apparent m.w. of 32,000, 22,000, and 6000, respectively. In addition, IgG antibodies recognized at least 17 other antigenic components. After subcellular fractionation, enrichment of the three major antigens recognized by IgM and IgG antibodies by the membrane fraction was observed. Solubilization of membrane-enriched preparations with a mixture of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that reacted with IgM or IgG antibodies. Treatment of Toxoplasma lysate preparations and various fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. Lipase treatment had no effect on the number or nature of antigens recognized by IgM antibody. Treatment with pronase and trypsin eliminated the 32,000 and 22,000 m.w. antigenic components detected by IgM antibodies, whereas such treatment had no effect on the 6000 m.w. component. Periodic acid-Schiff staining of polyacrylamide gels of Toxoplasma sonicates revealed the presence of three components corresponding to m.w. of 62,000, 45,000, and 6000, respectively. At least 15 components, including the 6000 m.w. component, directly bound concanavalin A.

Animals↗
PubMed1980

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.

Agglutination Tests↗
PubMed1977

[Cytochemical study of different stages in the life cycle of Toxoplasma gondii. I. Nucleic acids and proteins in endozoites].

Using the Feulgen technique in addition to the methyl green-pyronin and gallocyanin-chromalum staining, nucleic acids were detected in Toxoplasma gondii of strains SS-119 and RH DNA was revealed in the nuclei of both intracellular and free individuals examined on various days (2--6) after mouse inoculation. A high RNA content in the cytoplasm of endozoites is a most characteristic feature of this stage. However, no definite nucleolus has been demonstrated in the endozoite nucleus. Using the Fast green and Alcian blue techniques, resp., histones were detected in the endozoite nuclei whose locality corresponded to that of Feulgen-positive material. The Acrolein-Schiff method located aldehyde groups of protein in endozoites, the detected stuff being confined mainly to the nuclear and perinuclear areas of the parasite. Tannofilic protein seems to screen the endozoite body, no difference between nuclear and cytoplasmic staining being seen. Tryptophan and tyrosin were not detected in the endozoites of Toxoplasma. The results obtained on Toxoplasma endozoites are compared with the metabolic patterns seen in the host cells of the peritoneal exudate and with previous literature data.

DNA↗
PubMed1981

Toxoplasma-induced abortion in dairy goats.

Fifteen pregnant goats serologically negative for Toxoplasma gondii were inoculated orally with 10, 100, or 1,000 infective oocysts of the GT-1 strain of T gondii. Four goats received 10 oocysts, 6 received 100 oocysts, and 5 received 1,000 oocysts. Six pregnant goats served as noninoculated controls. All inoculated goats became pyretic (40-41 C) and were dyspneic between 4 and 12 days after inoculation (DAI). Two goats inoculated in early pregnancy (68 and 70 days) with 100 oocysts had enlarged uteri; fetal membranes but no fetuses were found when they were killed 86 and 88 DAI. Three goats aborted or had a stillborn kid 9, 10, and 11 DAI. The remaining 10 goats delivered kids infected with T gondii; 8 goats had at least 1 stillborn kid, and 2 goats had infected but otherwise normal kids at birth. Placenta and tissues of fetuses and kids were inoculated into mice for determination of Toxoplasma infectivity. Toxoplasma gondii was isolated from the fetal placenta of 6 of 7 goats as early as 10 DAI and as late as 62 DAI. The organism was isolated from every fetus or kid that was born 18 DAI but not from fetuses or kids born 9, 10, and 11 DAI. Two kids were born 15 DAI; T gondii was isolated from 1 kid but not from the other, suggesting that T gondii invaded fetal tissues between 11 and 15 DAI. Of the 20 transplacentally infected kids, T gondii was isolated more frequently from the skeletal muscle, heart, lung, and brain than from other tissues.

Abortion, Veterinary↗
PubMed1980

Lectin used in the purification process of Toxoplasma gondii tachyzoites.

Toxoplasma gondii tachyzoites were purified from mouse peritoneal exudates using lectins to remove the host cells. Best results were obtained with phytohemagglutinin P at a concentration of 0.01% (W/V), which provided a 99.7% pure Toxoplasma suspension. The process was reproducible and easy to perform. Toxoplasmas so obtained were infective and served as sources of high quality antigens for immunofluorescence, hemagglutination, and complement fixation tests.

Animals↗
PubMed1980

Toxoplasma gondii: membrane properties of active energy-dependent invasion of host cells.

The energy-dependence and the significance of membrane stability for the active invasion of host cells by Toxoplasma gondii has been examined in vitro. The penetration of Toxoplasma as measured by the infection rate can be significantly decreased by blocking the parasitic respiratory chain with CN-. The infection rate of blocked parasites is nearly normalized when glucose is present during the infection period. This suggests a strong energy-dependence for the active infection mechanism. DMSO (dimethylsulfoxide) known to increase membrane stability is able to lower the infection rate if either the target or the effector cell is pretreated. Ca2+ was shown to exhibit a similar effect. Procain, which destabilizes the cell membrane, stimulates the invasion of host cells by Toxoplasma parasites. The possible modes of action on the invasion schedule for the different effectors are discussed.

Animals↗
PubMedDate not supplied

[Isolation of "Toxoplasma gondii" membrane antigens using latex microspheres (author's transl)].

The authors describe a method for isolation of Toxoplasma gondii antigenic membrane components. Technical work is described into four points: 1) specific labelling of membrane proteins was realised by DD125ISA; 2) methacrylate microspheres were bound to unbroken toxoplasma membranes by the mean of an indirect technic with antibody molecules (double sandwich); these microspheres were used to modify the membrane density; 3) toxoplasmas bound to microspheres were broken by sonication; 4) microspheres fixed to membrane components were isolated by isopycnic ultracentrifugation on continuous sucrose gradient.

Antigens, Surface↗
PubMed1981

[Natural adaptation of the white rat to Toxoplasma gondii].

White rats were found to be resistant to 3 x 10(7) Toxoplasma gondii organisms (RH strain) inoculated either sc or ip. That resistance was age-dependent since survival time of 1- or 5-day-old rats inoculated with 10(4) or 10(6) tachyzoites was lower as compared with those 10 or 15 days old. Organisms kept in contact with a lysate of white rat complete peritoneal exudate showed reduced capacity to infect mice. Peritoneal exudate of 5-day-old rats showed a stronger effect than that of 1, 2, 3, or 4 day-infected animals. After 10 days the anti-toxoplasma effect diminished and it almost disappeared after 15 days of infection. Apparently age and macrophage activity are very important factors in the natural adaptability of the white rat to Toxoplasma gondii.

Adaptation, Biological↗
PubMed1978

A method of mass cultivation of Toxoplasma gondii in cell culture.

Large quantities of Toxoplasma gondii were cultivated in human larynx carcinoma cells (H.Ep 2, heteroploid). Virulent Toxoplasma gondii, strain RH, which were obtained from the peritoneal exsudate of mice 2 days after infection served as inoculum. As culture medium for the cells and for growth medium, Minimum Essential Medium (MEM) supplemented by 5% foetal calf serum and 200,000 IE/1 Penicillin plus 20 mg/1 Sulmycin was used. 2--3 days after inoculation, 150--200 times the amount of the inoculated Toxoplasmas could be harvested in one or several harvests.

Cells, Cultured↗
PubMed1995

Sources and reservoirs of Toxoplasma gondii infection on 47 swine farms in Illinois.

Field studies were conducted on 47 swine farms in Illinois during 1992 and 1993 to identify sources and reservoirs of Toxoplasma gondii infection. Blood samples were obtained from swine and from trapped wildlife. Serum antibodies to T. gondii were determined using the modified agglutination test, incorporating mercaptoethanol. Antibodies to T. gondii (titer > or = 25) were found in 97 of 4,252 (2.3%) finishing pigs, 395 of 2,617 (15.1%) sows, 267 of 391 (68.3%) cats, 126 of 188 (67.0%) raccoons, 7 of 18 (38.9%) skunks, 29 of 128 opossums (22.7%), 6 of 95 (6.3%) rats, 3 of 61 (4.9%) white-footed mice (Peromyscus sp.), and 26 of 1,243 (2.1%) house mice (Mus musculus). Brains and hearts of rodents trapped on the farm were bioassayed in mice for the presence of T. gondii. Toxoplasma gondii was recovered from tissues of 7 of 1,502 (0.5%) house mice, 2 of 67 (3.0%) white-footed mice, and 1 of 107 (0.9%) rats. Feces of 274 cats trapped on the farm and samples of feed, water, and soil were bioassayed in mice for the presence of T. gondii oocysts. Toxoplasma gondii was isolated from 2 of 491 (0.4%) feed samples, 1 of 79 (1.3%) soil samples, and 5 of 274 (1.8%) samples of cat feces. All mammalian species examined were reservoirs of T. gondii infection. All farms had evidence of T. gondii infection either by detection of antibodies in swine or other mammalian species, or by detection of oocysts, or by recovery from rodents by bioassay. The possibility of transmission of T. gondii to swine via consumption of rodents, feed, and soil was confirmed.

Animal Feed↗
PubMed1978

[Experimental studies on the affinity of Toxoplasma gondii to various organs of mice (author's transl)].

In response to their alleged "uterus affinity" the organotropism of various Toxoplasma strains was investigated in the female mouse. Using quantitative histological (immunofluorescence) and serological (Sabin-Feldman-dye test, complement fixation test), we examined the brain, heart, lung, spleen, striated muscle, uterus and ovaries. The following results were obtained: All four of the Toxoplasma strains (ALT, DX, WEISS, GAIL) showed a definite neurotropism. Located in the brain were one hundred times as many cysts as in the sexual organs and ten times as many as in the reamining organs. The uterus and ovary show a low susceptibility, invasion rarely occuring. An "uterus affinity" could not be confirmed. All four Toxoplasma strains showed the same affinity for the various organs. The strain ALT showed the most virulence and formed the most cysts.

Animals↗
PubMed1995

Acquired retinochoroiditis in hamsters inoculated with ME 49 strain Toxoplasma.

PURPOSE: These studies were undertaken to establish an animal model for use in studies of ocular toxoplasmosis. An animal model is needed to examine the development, progression, and resolution of ocular Toxoplasma infections and to study the effects on the disease of currently used and experimental therapies. METHODS: Cysts of the ME 49 strain of Toxoplasma gondii were injected intraperitoneally into each of 60 golden hamsters. The hamsters' eyes were examined before inoculation and at intervals after inoculation, and fundus photographs were taken. Histologic sections were analyzed and photographed to document the ocular effects of the infection. RESULTS: Retinochoroiditis was found in both eyes of all hamsters within 2 to 3 weeks of inoculation. The disease resolved spontaneously without treatment and was quiescent in most cases at 12 weeks after inoculation. The animals remained in good general health, and those tested had high antibody titers to Toxoplasma (1:256 to 1:32,000) at 6 months after the infection. The discovery of cysts and lesions in the retina confirmed the diagnosis. CONCLUSIONS: Although the lesions were not identical to those of human disease, this animal model of ocular toxoplasmosis offers several advantages: reproducibility, short incubation time, spontaneous resolution without treatment, consistent production of cysts, and ease of inoculation intraperitoneally without intraocular injection.

Animals↗
PubMed1995

Detection of Toxoplasma gondii-specific IgA in the aqueous humor of cats.

Toxoplasma gondii-specific IgA, IgM, and IgG were measured by ELISA in the serum and aqueous humor of 29 client-owned cats with endogenous uveitis and 7 specific-pathogen-free cats tested sequentially for 20 weeks after inoculation with T gondii. Local antibody production in aqueous humor was estimated by multiplying the aqueous humor-to-serum T gondii-specific antibody ratio by the serum-to-aqueous humor total IgG (C value) or calicivirus-specific IgG (CTC value) ratio. Evidence for local production of antibody in aqueous humor was defined as C value greater than 8 or CTC value greater than 1. Toxoplasma gondii-specific IgM CTC values, IgG CTC values, or IgA CTC values greater than 1 were detected in the aqueous humor of 18 of 29 (62.1%) client-owned cats with endogenous uveitis; 2 cats had IgA CTC values greater than 1 without detectable IgM or IgG in aqueous humor. Toxoplasma gondii-specific IgM was not detected in the aqueous humor of experimentally inoculated cats before or after inoculation. Immunoglobulin G C values greater than 8 were detected in all 7 experimentally inoculated cats and ranged from 10.4 to 145.5. Immunoglobulin G C values greater than 8 were first detected 4 to 8 weeks after T gondii inoculation and were undetectable by week 16 after inoculation. Immunoglobulin A C values greater than 8 were detected in 4 of 7 cats and ranged from 12.7 to 264.3. Immunoglobulin A C values greater than 8 were first detected 4 to 8 weeks after inoculation, and were detected in 2 cats during week 20 after inoculation. It was concluded that some cats infected with T gondii develop detectable concentrations of T gondii-specific IgA in aqueous humor.

Animals↗
PubMed1995

Identification of two protein serine/threonine kinase genes and molecular cloning of a SNF1 type protein kinase gene from Toxoplasma gondii.

We report for the first time the identification of two protein kinase genes, in Toxoplasma gondii. Based on the conserved amino acid sequence motifs of catalytic subdomains VI(b) and IX of known protein serine/threonine kinases, degenerate oligonucleotides were synthesized and used in PCR to produce 196 and 373 bp DNAs. They encoded stretches of amino acid sequences characteristic of protein serine/threonine kinases. Dot and Southern blot analysis confirmed that these PCR products were of Toxoplasma origin. Screening of a genomic library of the organism with the 196 bp PCR product as a probe yielded 3 ToxPK1 g clones. Nucleotide sequence of two of these clones, revealed that the protein encoded, TOXPK1 resembled other carbon catabolite derepressing regulatory protein kinases. Therefore, we suggest that TOXPK1 could play a role in the interconversion of active and passive life-cycle stages of this parasite. RT-PCR studies on Toxoplasma tachyzoites' total RNA suggested that ToxPK1 gene is developmentally regulated. The 373 bp PCR product, however encoded a polypeptide that resembled the catalytic subunit of other cAMP-dependent protein kinases. Hence, this protein (TOXPK2) was considered as a product of another gene, ToxPK2.

Amino Acid Sequence↗
PubMed1994

Toxoplasma gondii oral infection induces specific cytotoxic CD8 alpha/beta+ Thy-1+ gut intraepithelial lymphocytes, lytic for parasite-infected enterocytes.

The first line of defense of the host after primary infection by Toxoplasma gondii is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IEL) (mainly of CD8 phenotype), mucus, and secretory Igs. T. gondii cysts were administered orally to CBA/J mice to determine whether parasite-specific cytotoxic IEL can be elicited within the intestine. We found that oral infection led to an increase in the relative percentages of the CD8 beta + and Thy-1+ IEL populations between day 9 and day 13 after infection. At these times, T. gondii-primed, but not control mice, generated parasite-specific cytotoxic effector IEL for toxoplasma-infected macrophages. This cytotoxic activity was genetically restricted. By using magnetically activated cell sorting, the effector IEL were shown to be CD8 alpha/beta + Thy-1+ T lymphocytes and FACScan analysis revealed that they mainly express TCR-alpha beta. A significant level of cytotoxicity was also observed against T. gondii-infected epithelial cells from the MODE-K cell line, suggesting that parasite-specific IEL may ensure epithelial integrity by rapidly killing infected enterocytes. Finally, toxoplasma stimulation in vitro led to IFN-gamma production by T. gondii-primed IEL. Taken together, these data suggest that antigen-specific IEL, bearing the CD8 alpha/beta + Thy-1+, TCR-alpha beta + phenotype, can act directly as effector cells through a specific CTL activity at the intestinal level and may indirectly activate other mucosal effector mechanisms through IFN-gamma secretion.

Administration, Oral↗
PubMed1994

Toxoplasma gondii infection in marrow transplant recipients: a 20 year experience.

Twelve of 3803 consecutive marrow allograft patients treated at this center over the past 20 years have had a post-transplant tissue diagnosis of toxoplasmosis: 10 at autopsy and 2 by brain biopsy. This infection was identified in none of 509 autologous marrow recipients. Occurrence of toxoplasmosis was 0.31 cases per 100 allogeneic transplants and 1.0 per 100 autopsies. An estimated 15% of allogeneic transplant recipients were seropositive for Toxoplasma gondii and 2% of seropositive patients developed toxoplasmosis. Pre-transplant serology was positive by both dye and agglutination tests in 11 infected patients tested. Sequential IgG, IgM, IgA, IgE antibody titers to T. gondii and the differential agglutination ratio were not helpful in diagnosing toxoplasmosis. Median day of clinical presentation was day 59 post-transplant (35-97 days) and of diagnosis, day 62 after transplant (37-143 days). Eleven patients had graft-versus-host disease (GVHD) of grades II-IV. All 12 patients died. Infection was diagnosed prior to death in only 16% of patients and contributed to death in at least 40%. Histopathology revealed tachyzoites of T. gondii most prevalent in brain (100%), heart (67%) and lungs (33%), and toxoplasma cysts alone in heart (33%) and lungs (22%). Toxoplasma infection was diagnosed in two patients receiving trimethoprim-sulfamethoxazole for Pneumocystis carinii pneumonia prophylaxis suggesting this was insufficient prophylaxis for toxoplasmosis. Toxoplasmosis appeared to occur by reactivation within the first 6 months after marrow transplant. Infection developed in patients who were seropositive for T. gondii pre-transplant, had received allogeneic marrow and had severe GVHD.

Adolescent↗