Transgenic studies of peripheral and central glia

This paper reviews recent studies on the life cycle of Toxoplasma gondii. Tachyzoites, bradyzoites, and sporozoites are the three infectious stages of T. gondii. Humans and animals become infected mainly by ingesting bradyzoites or oocytes. After ingestion, both bradyzoites and sporozoites convert to tachyzoites inside tissues. The conversion of tachyzoites to bradyzoites and bradyzoites to tachyzoites is of biological and clinical significance because bradyzoites are less susceptible to chemotherapy and reactivation of bradyzoites to tachyzoites is considered the cause of fatal toxoplasmosis in AIDS patients. Of all the methods currently available to assess stage conversion of T. gondii, feeding infective stages to cats is the most reliable method. Felidae, the definitive hosts of T. gondii excrete oocysts 3-10 days after ingesting tissue cysts/bradyzoites, > or = 18 days after ingesting oocysts, and > or = 13 days after ingesting tachyzoites.     

Sex, Task, and Behavioral Flexibility Effects on Leadership Perceptions

We critically review and summarize information on the prevalence of Toxoplasma gondii infections in rats, mainly Rattus norvegicus, and their possible role as a source of infection for larger carnivores and omnivores. We also review information on immunology and natural resistance, contributing to the model value of rats in the analysis of human infection. Rats can be successfully infected with oocysts (sporozoites), tissue cysts (bradyzoites), and tachyzoites. Even adult rats, that are resistant to clinical toxoplasmosis, can be infected orally with a few oocysts or tissue cysts. Infections with tachyzoites of the RH strain are highly variable. Congenital transmission of T. gondii occurs at a high rate when rats are infected during pregnancy. Congenitally infected rats can harbor viable T. gondii in the absence of detectable antibodies to T. gondii and rats with low antibody titers may harbor few or no organisms. The isolation of viable T. gondii by bioassay is the only reliable means to determine persistence of chronic T. gondii infection in feral rats. No evidence was found for maintenance of T. gondii in rats by vertical transmission in the absence of cats.     

Caprine toxoplasmosis: abortion, clinical signs, and distribution of Toxoplasma in tissues of goats fed Toxoplasma gondii oocysts

Thirteen goats (9 does and 4 bucks) were each inoculated orally with 10,000 infective Toxoplasma gondii oocysts. Three does and one buck were used as noninoculated controls. In 2 to 4 days after inoculation (DAI), inoculated goats became dull, pyrectic (40 to 41 C), and anorectic. Three goats died (10, 10, and 14 DAI) and two goats were killed (7 and 32 DAI) because they were moribund; also, 3 does aborted, 2 had weak kids, and 2 had dead fetuses. Toxoplasma was isolated from the placenta of three goats, and the fetal tissues of four goats. The control goats remained asymptomatic. The distribution of T gondii in blood and other tissues was studied by inoculation of mice with caprine tissues. Parasitemia was detected in 7 of 7 goats--beginning 4 DAI in 1 goat, 5 DAI in 5 goats, and 8 DAI in 1 goat. The parasitemia lasted 3 to 10 days. Toxoplasma was isolated from the milk of 2 goats at 12 and 14 DAI. Toxoplasma was isolated from 15 or more tissues of 5 goats killed 7 to 35 DAI and from 10 tissues of 2 goats killed 69 and 95 DAI.     

Experimental toxoplasmosis in broiler chicks

To evaluate chicken toxoplasmosis both as an economic and a public health subject, 84 broiler chicks of a commercial strain, 30 days old, were distributed into seven groups of 12 birds (three replications of four chicks) experimentally infected with three developing T. gondii stages of the P strain as follows: tachyzoites, intravenous (two groups: 5.0 x 10(5) and 5.0 x 10(6)), cysts, per os (two groups: 1.0 x 10(2) and 1.0 x 10(3)) and oocysts, per os (three groups: 5.0 x 10(2), 5.0 x 10(3) and 5.0 x 10(4)). Twelve chicks received only a placebo (control group). During the next 30 days the following parameters were estimated: productivity (weight gain and feed conversion), clinical signs, including rectal temperature and parasitemia (bioassay). No clinical signs suggesting toxoplasmosis were seen and no statistical differences on productivity standards were found in comparison between inoculated and control chicks. However, fowls inoculated with tachyzoites and oocysts occasionally showed hyperthermia. Some haematological changes were detected in fowls inoculated with T. gondii. Anatomo-histopathological changes were not observed. From 14 parasitemias detected, 35.7% appeared on the 5th day after inoculation and 57.1% of them resulted from oocysts inoculation. After 30-35 days all birds were slaughtered: fragments from 12 organs or tissues from each of them were subjected to artificial peptic digestion and after that injected into T. gondii antibody-free mice (IIFR). T. gondii was detected in brain (12), pancreas (five), spleen (five), retina (five), kidney (two), heart (four), proventriculus (three), liver (two), intestine (two), lung (one), and skeletal muscle (one). Similar to observations with parasitemia, from 42 T. gondii isolations, 59.5% came from chicks which had received oocysts. It can thus be inferred that the developing form, expelled by cats, is the most important for T. gondii chicken infection and that brain is the most infected organ in birds. Attention must be paid to the potential importance of chicken meat in public health, since T. gondii was isolated from skeletal and heart muscles.     

Novel anticoagulants based on direct inhibition of thrombin and factor Xa

Decoquinate is an anticoccidial agent that inhibits respiration in the parasites mitochondrion. We examined human foreskin fibroblast cell cultures infected with the normally tissue cyst-less RH strain of Toxoplasma gondii and treated with decoquinate for evidence of tissue cyst induction and formation. Transmission electron microscopy observations demonstrated tissue cysts in decoquinate-treated cultures on days 3, 4, 5, and 6 after inoculation. Tissue cysts contained a tissue cyst wall that enclosed stages that resembled tachyzoites and stages that were structurally bradyzoites. Similar treatment of human foreskin fibroblast cells infected with tachyzoites of the TS-4 temperature-sensitive mutant of the RH strain did not result in production of tissue cysts.     

Antibody responses measured by various serologic tests in pigs orally inoculated with low numbers of Toxoplasma gondii oocysts

OBJECTIVE: To follow antibody responses measured by various serologic tests in pigs orally inoculated with low (< or = 10 oocysts) numbers of Toxoplasma gondii oocysts. ANIMALS: 24, 2- to 3-month-old pigs. PROCEDURE: Pigs (n = 42) were inoculated orally with 10 (14 pigs) or 1 (28 pigs) infective oocysts, and 6 pigs served as uninoculated controls. Blood (serum) samples were obtained at 1- to 3-week intervals until euthanasia. At necropsy, the brain, heart, and tongue of pigs were bioassayed in mice and cats for isolation of T gondii. Modified agglutination test (MAT), using whole, fixed tachyzoites and mercaptoethanol; latex agglutination test (LAT); indirect hemagglutination test (IHAT); Sabin-Feldman dye test (DT); and ELISA were used to evaluate serologic responses to T gondii. RESULTS: T gondii was isolated from tissues of 13 of 14 pigs each fed 10 oocysts, 17 of 28 pigs each fed 1 oocyst, and 0 of 6 control pigs. 29 of 30 T gondii-infected pigs developed antibodies when measured by MAT, DT, and ELISA; the 1 seronegative-infected pig had been fed 10 oocysts and was euthanatized 69 days after inoculation. LAT detected antibodies in 26 of 30 T gondii-infected pigs. IHAT detected antibodies in 11 T gondii-infected pigs. CONCLUSION: MAT, DT, and ELISA were more sensitive serologic assays than LAT and IHAT for detecting antibodies induced by low numbers of T gondii in pigs.     

Immunization with E. coli produced recombinant T. gondii SAG1 with alum as adjuvant protect mice against lethal infection with Toxoplasma gondii

Polyclonal rabbit antibodies against recombinant Toxoplasma gondii SAG1 antigen expressed in E.coli recognize T. gondii and the antibodies significantly reduced T.gondii adherence and/or invasion into the host cell as did a monoclonal antibody against a conformational epitope of the SAG1 antigen. Groups of outbread NMRI mice were immunized with recombinant T. gondii SAG1 antigen in alum. The antibody response to immunizations was dominated by a Th2 response with production of T.gondii specific IgG1 antibodies. Challenge with tachyzoites from the virulent RH-strain produced a Th1 response dominated by the production of specific IgG2a antibodies and moderately boosted the IgG1 response, and challenge with bradyzoites from the avirulent SSI119-strain showed the same pattern. Immunization with rSAG1 resulted in a significant increased survival after challenge with tachyzoites of the RH-strain. Immunization with E.coli expressed recombinant SAG1 in alum induce partial protective immunity against lethal infection with T. gondii in mice.     

Murine Th1 clone defines a 60 kD tachyzoite antigen of Toxoplasma gondii

Toxoplasma gondii-specific murine CD4+ T cell clone 3Tx9 belongs to the Th1 subtype by virtue of secreting high levels of interleukin(IL)-2, interferon-gamma and tumor necrosis factor without producing IL4 and IL10. To characterize the clonal antigen, Toxoplasma lysate-was separated by SDS-PAGE and probed in T cell blot analysis with 3Tx9 T cells, revealing a fraction of about 60 kD molecular weight. This fraction proved highly stimulatory also for CD4+ splenocytes isolated from infected mice. The expression pattern of the relevant 60 kD antigen was determined by challenge of clone 3Tx9 with T. gondii strains from all three intraspecies subgroups and tachyzoites versus bradyzoites isolated from two strains as a source of antigen. While the T cell clone reacted with tachyzoites of all strains tested, bradyzoites lacked antigenic activity. Parallel T cell blot and ELISA confirmed co-migration of the T cell-stimulatory antigen p60 and rhoptry proteins ROP1, ROP2,3,4, and ROP5 among which ROP1 is a molecule of similar size and has only been shown on tachyzoites. However, a ROP1 knock-out Toxoplasma mutant still had antigen activity for 3Tx9 T cells. Since the two known tachyzoite-specific proteins, surface antigens SAG1/p30 and SAG2/p22, have a much lower molecular weight, we suggest that p60 represents a new T. gondii tachyzoite marker which is defined by clone 3Tx9.     

Cyst formation by Toxoplasma gondii in vivo and in brain-cell culture: a comparative morphology and immunocytochemistry study

Formation of Toxoplasma gondii cysts was examined in cultured murine brain cells and was compared with the development of cysts in mouse-brain tissue. Cultures of mixed glial cells from neonatal mouse brain were infected with bradyzoites of the avirulent T. gondii strain DX. The development and maturation of Toxoplasma cysts was monitored for up to 63 days after inoculation. Transmission electron microscopy indicated that in-vitro-derived cysts were morphologically similar to tissue cysts and were located intracellularly, even for up to 63 days postinfection. For immunohistological and immunocytochemical examination of both in-vivo- and in-vitro-infected material, monoclonal antibody (mAb) CC2 was used. MAb CC2 was shown to detect specifically the underlying granular material of the cyst wall without binding to the limiting membrane of the parasitophorous vacuole. This reactivity of mAb CC2 allows the distinction of bradyzoite-containing cysts from parasitophorous vacuoles harboring tachyzoites both in vitro and in vivo.     

Sequential reactivity of serum against cyst antigens in Toxoplasma infection

AIMS: To compare the recognition of Toxoplasma gondii tachyzoites and cysts by sera, from 10 patients. METHODS: Recognition of antigens from purified tachyzoites (RH strain) and bradyzoites (18691 strain) was compared using western immunoblotting. Sequential serum samples from 10 patients and one laboratory acquired RH infection were used. RESULTS: Recognition of cyst antigens was relatively low and occurred late in infection. The two stages were antigenically distinct with only a few shared bands. CONCLUSION: Immunological recognition of the cystic stage of T gondii is low. This implies that either cysts are poorly immunogenic or that cyst antigen is not available for processing and presentation.     

Comparative infectivity of Toxoplasma gondii bradyzoites in rats and mice

Infectivity of Toxoplasma gondii bradyzoites was compared in outbred female Sprague Dawley rats and outbred Swiss Webster mice. Rats inoculated subcutaneously with 1-10 bradyzoites of the 2 strains of T. gondii (VEG and GT-1) developed persistent infection, whereas an infective dose by the oral route was 10-1,000 bradyzoites. The infectivity of bradyzoites of the VEG and the GT-1 strains of T. gondii in rats by the subcutaneous route was comparable to that in mice.     

Fas-FasL interaction involved in pathogenesis of ocular toxoplasmosis in mice

Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation of Toxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8(+) and CD4(+) T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000 T. gondii tachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8(+) than CD4(+) T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lpr and B6-gld mice than in wild-type B6 mice (P < 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.     

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.     

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.     

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.     

Loss of stages after continuous passage of Toxoplasma gondii and Besnoitia jellisoni

Toxoplasma gondii, passed from mouse to mouse in the tachyzoite stage for 30-35 generations, developed cysts, which when fed to cats, failed to produce oocysts. Besnoitia jellisoni, passed similarly for 20 generations, lost the capacity to form cysts. These phenomena are explained by a loss of genomes or gene products during the rapid passage selecting for tachyzoites.     

Diagnosis of toxoplasma abortion in ewes by polymerase chain reaction

Eighteen oestrus-synchronised ewes were infected experimentally with 1500 sporulated oocysts of Toxoplasma gondii between 80 and 90 days of gestation. The infection induced pyrexia and specific antibody in all the ewes. One ewe resorbed its fetus, five ewes aborted and 12 delivered live, congenitally-infected lambs whose pre-colostral serum was antibody-positive. Tissues from the aborted fetuses and placentae from the live lambs were examined for toxoplasma infection by polymerase chain reaction (PCR) amplification of the B1 gene and by mouse inoculation. Using a simple protocol of tissue preparation without DNA extraction and a nested format, PCR was as sensitive as mouse inoculation. Placental cotyledon gave a higher sensitivity of detection than brain, lung or liver, and 16 of 19 placentae were positive by PCR compared with 13 of 18 by mouse inoculation. In mock-infected tissues, as few as 10 tachyzoites could be detected. The PCR could be applied to tissues unfit for mouse inoculation.     

Remarks of Louis W. Sullivan, MD, before the annual AIMBE meeting

Treatment with pyrimethamine and sulfamethoxazolum combination administered via intragastric route, was started 2 hours after each mouse received an intraperitoneal injection of a lethal inoculum of 5 x 10(3) tachyzoites of RH strain toxoplasma gondii and the therapy was lasted for 52 days. Combined therapy of the two drugs resulted in 83.3% survival rate in mice. PCR and the conventional mouse inoculation (MI) were compared for their validity as an curative effect assessing method. The positive rates of PCR and MI were 63.64% and 18.18%, respectively. The results suggest that PCR can be used in studies involving drug screening and curative effect assessment of anti-Toxoplasma chemotherapy in vivo. In addition, the results indicated that longer course of treatment against toxoplasmosis is required in clinical practice.     

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.     

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in sera from camels from Egypt

Sera from camels from Egypt were examined by the direct agglutination tests incorporating mercaptoethanol for antibodies to Neospora caninum and Toxoplasma gondii. Antibodies to N. caninum were found in 6 of 161 camels in titers of 1:40 (2 camels) and 1:80, 1:160, 1:640, and 1:1280 in 1 camel each, using N. caninum formalin preserved whole tachyzoites as antigen. Antibodies to T. gondii were found in 17.4% of 166 camels in titers of 1:25 (3 camels), 1:50 (18 camels). and > 1:500 (8 camels) using T. gondii tachyzoites. All 6 camels with N. caninum antibodies had no T. gondii antibodies in 1:4 dilution of serum, indicating specificity of the reaction. This is the first report of N. caninum prevalence in Egypt.