A heterophile antigen associated with experimental toxoplasmosis

The biological characteristics of a heterophile protein (HP) in peritoneal exudate from mice, hamsters, rats, and guinea pigs infected with Toxoplasma gondii were studied by immunofluorescence, immunoelectrophoresis and immunodiffusion techniques using specific antisera raised in rabbits. HP of mice had the highest antigenicity, HP of hamsters and rats had intermediate antigenicity and HP of guinea pigs had the lowest antigenicity. HP was found in normal peritoneal exudates from mice, hamsters and rats inoculated with paraffin oil instead of T. gondii and in normal guinea pig serum. HP was detected by the fluorescent antibody technique on the surface of T. gondii in peritoneal exudates of mice, but not on mouse peritoneal cells, and by the indirect fluorescent antibody technique on L cells infected with T. gondii and on free Toxoplasma derived from them, but not on uninfected L cells. T. gondii could make host cells produce HP to cover its surface for protection. The relation between HP from host cells and T. gondii is discussed.     

Absence of vacuolar membrane involving Toxoplasma gondii during its intranuclear localization

Tachyzoites of Toxoplasma gondii were located inside the nucleus of both skeletal muscle cells infected in vitro and peritoneal exudate cells collected from infected mouse in vivo. Ultrastructural analysis demonstrated that T. gondii invades the nucleus of host cells by the parasite apical region and with constriction of its body. We noted that the rhoptry, a secretory organelle of the parasite that is involved in the host cell invasion mechanism, was empty in the intranuclear T. gondii. The parasites were found in the nuclear matrix without evidence of the vacuolar membrane. Frequently, new parasites invaded host cell nucleus, which was already infected. The significance of this nuclear invasion could reflect an alternative route of T. gondii for its transitory survival or an escape mechanism from the host immune response during the in vivo infection (or both).     

Roles of Toxoplasma gondii-derived heat shock protein 70 in host defense against T. gondii infection

C57BL/6 mice receiving intraperitoneal injection of Toxoplasma gondii -derived heat shock protein 70 (T.g. HSP70) on day 3 post T. gondii infection succumbed by day 9 post infection, while vector protein-injected control mice survived more than 6 months. The deteriorating effect of T.g. HSP70 on host immune responses was dose-dependent. By T.g. HSP70 injection, T. gondii loads increased in various organs of T. gondii-infected mice. Th2 cytokines such as IL-4 and IL-10 were continuously produced from spleen and peritoneal exudate cells of T. gondii -infected mice by injection of T.g. HSP70. Furthermore, nitric oxide production from peritoneal macrophages in T. gondii-infected mice was reduced by T.g. HSP70.     

A soluble phospholipase of Toxoplasma gondii associated with host cell penetration

We previously reported that phospholipase increases host cell penetration by Toxoplasma gondii. Here we show that calcium-dependent phospholipase A (PLA) activity is found in the supernatant of sonically disrupted T. gondii. When fractions of disrupted T. gondii were incubated with host cells, the release of fatty acids and lysolipids was detected. Fractions of sonically disrupted T. gondii with PLA activity increased T. gondii host cell penetration in a bioassay. In addition, a protein of approximately 20 kDa was detected by immunoblot of T. gondii antigens with horse antiserum to snake venom, the major antibody of which recognizes PLA2. Incubation of T. gondii with exogenous PLA2 resulted in increased solubility of a rhoptry protein. This protein, which we previously characterized as involved with enhanced parasite invasion of host cells and which is recognized by monoclonal antibody Tg49, was detected in increased amounts in supernatant fractions of extracellular parasites treated with PLA2. Whereas without PLA2 treatment, it is only slightly soluble under physiological conditions. This raises the possibility that PLA may be implicated in the release of rhoptry proteins.     

Rat macrophage activity against Toxoplasma gondii studied by electron microscopy

An electron microscope model was used to study the effect of rat peritoneal macrophages on Toxoplasma gondii. 10(7) tachyzoites were injected i.p. in 30 days-old rats. After 1, 2, 4, 8 and 24 h peritoneal exudate was withdrawn and infected phagocytic cells were prepared for electronic microscope studies. Toxoplasma organisms inside of rat macrophages showed remarkable lesions such as vacuolization and organisms were totally lysed inside of macrophages of more than 8 h infection rats. The results confirm at molecular level, the importance of rat macrophages in the natural adaptation of this rodent to T. gondii.     

A novel B-2 suppressor cell regulating susceptibility/resistance of mice to Toxoplasma gondii infection

Irradiation treatment enhanced resistance of C57BL/6, but not BALB/c against Toxoplasma gondii infection. Six Gy-irradiated (IR) C57BL/6 recipients of B-2 cells from T. gondii-infected C57BL/6 died after infection. B-2 suppressor cells from infected C57BL/6 enhanced production of IL-4 and IL-10 in peritoneal exudate cells (PECs), and down-regulated NO release in peritoneal macrophages after infection. On the other hand, B-2 suppressor cells were not detected in a strain, BALB/c, resistant against infection. These data indicated that irradiation-sensitive B-2 cells regulated susceptibility/resistance in mice against T. gondii infection.     

Use of mouse macrophage cell lines for in vitro propagation of Toxoplasma gondii RH tachyzoites

In most laboratories, Toxoplasma gondii is maintained in mice and is studied in vitro using nonlymphoid cell lines or primary mouse macrophages. In this study, three rapidly dividing mouse macrophage cell lines (J774 A.1, P388D1, RAW264.7) were evaluated for their suitability for studying the RH strain of T. gondii. For comparison, tachyzoites were also grown in two slowly dividing epithelial cell types: a rat lung cell line (L2) and a bovine turbinate cell line (BT). Various inocula of T. gondii were added to the above cells and tachyzoites were harvested from the culture supernatants after 2-8 days of infection. The mouse macrophage cell lines supported rapid growth of T. gondii RH allowing up to a 300-fold increase of the inoculum in 2-4 days. L2 and BT supported slower growth of T. gondii (10- to 90-fold increase of inoculum in 5 to 8 days) and, thus, may be more suitable for assessment of host cell-parasite interactions and drug activity. Toxoplasma gondii RH isolated from each of the cell cultures described were able to multiply in all cell types used. Protein profiles of whole tachyzoite isolated from mice or cell cultures and protein profiles of the corresponding soluble and membrane fractions of the intraphagosomal membrane network were similar as seen after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In mice, intraperitoneal injection of 10(6), 10(5), and 10(3) tachyzoites isolated from the cell cultures or from infected mice caused death after 4, 5, and 8 days, respectively, indicating that parasites grown in vitro retained virulence.     

A Soluble Phospholipase of Toxoplasma gondii Associated with Host Cell Penetration

We previously reported that phospholipase increases host cell penetration by Toxoplasma gondii . Here we show that calcium-dependent phospholipase A (PLA) activity is found in the supernatant of sonically disrupted T. gondii . When fractions of disrupted T. gondii were incubated with host cells, the release of fatty acids and lysolipids was detected. Fractions of sonically disrupted T. gondii with PLA activity increased T. gondii host cell penetration in a bioassay. In addition, a protein of approximately 20 kDa was detected by immunoblot of T. gondii antigens with horse antiserum to snake venom, the major antibody of which recognizes PLA₂. Incubation of T. gondii with exogenous PLA₂ resulted in increased solubility of a rhoptry protein. This protein, which we previously characterized as involved with enhanced parasite invasion of host cells and which is recognized by monoclonal antibody Tg49, was detected in increased amounts in supernatant fractions of extracellular parasites treated with PLA₂. Whereas without PLA₂ treatment, it is only slightly soluble under physiological conditions. This raises the possibility that PLA may be implicated in the release of rhoptry proteins.     

Deficiency in the anti-apoptotic protein A1-a results in a diminished acute inflammatory response

A1 is an anti-apoptotic member of the Bcl-2 family that is up-regulated in inflammatory myeloid cells. In the present study, we investigated the role of A1 in the maintenance of acute inflammation in mice. Mice possess three genes encoding highly related isoforms of A1. A1-a isoform mRNA was minimally expressed in resident peritoneal macrophages, but was present at a 300-fold higher level in inflammatory macrophages elicited by i.p. infection with Toxoplasma gondii. In comparison, A1-b and A1-d levels were 3- and 10-fold higher, respectively. Peritoneal leukocytosis was decreased in infected A1-a-deficient mice compared with wild-type, and this reduction was associated with a small but reproducible enhancement of survival. These effects could not be explained by an alteration in peritoneal parasite load, nor by increased apoptosis of infected inflammatory cells, which were protected from cell death by an A1-a-independent mechanism. Increased apoptosis in inflammatory neutrophils was observed sporadically in A1-a-deficient mice. Regulation of apoptosis by A1-a may be an important proinflammatory event in acute host responses.     

Preservation of laboratory strains of Toxoplasma gondii in vitro

A procedure to obtain viable stabilates of virulent laboratory strains of Toxoplasma gondii with a prolonged storage life is described. Viable endozoites recovered from the sediment of mouse exudate or tissue cultures (LEP, HeLa) are suspended in Eagle's MEM medium supplemented with 10% calf serum and 10% dimethylsulfoxide and sealed into glass ampoules of 1-2 ml in volume. The ampoules are placed in an apparatus for gradual cell freezing, frozen to --35 degrees C at a rate of --1 degree C/min, and stored in liquid nitrogen. Reinoculation experiments on mice given the suspension intraperitoneally confirmed that such Toxoplasma gondii strains retain viability for at least 4 years. This in vitro preservation technique is compared with the analogous T. gondii preservation procedures described in the literature.     

Pathogenicity of Toxoplasma gondii through B-2 cell-mediated downregulation of host defense responses

IFN-gamma is the primary mediator of anti-parasite effector mechanisms against Toxoplasma gondii. After intraperitoneal infection with the Fukaya strain of T. gondii, unirradiated IFN-gamma knock-out (GKO) mice transferred with wild type (WT) CD8+ effector T cells from infected mice failed to induce the production of IFN-gamma and died, whereas irradiated (IR) GKO mice transferred with WT CD8+ T cells induced IFN-y production and survived more than 6 months. IR GKO mice transferred with WT CD8+ T cells together with GKO B-2 cells died 8 days after infection, whereas those transferred with WT CD8+ T cells together with B-la or T cells survived. B-2 cells of infected GKO mice activated CD11b+ cells for IL-4 production, and down-regulated NO release, STAT1 phosphorylation, and interferon regulatory factor-1 expression in the peritoneal exudates cells of IR GKO mice transferred with WT CD8+ T cells together with GKO B-2 cells after infection. Thus, B-2 cells in T. gondii-infected mice act as suppressor cells in the host defense of infected mice.     

Cold stress-induced modulation of cell immunity during acute Toxoplasma gondii infection in mice.

Infection with Toxoplasma gondii in the acute phase results in nonspecific suppression of immunologic function in mice and humans. The present study examined the effects of a physical stressor, i.e., cold stress (CS), on macrophage function (nitrite production, parasite survival) and splenic blastogenesis in the acute phase of murine T. gondii infection. In our stress paradigm, female BALB/c mice were placed in cold water (1 +/- 0.5 C), 5 min each day for 8 days. Nitrite production and parasite survival were measured in cultured peritoneal macrophages obtained from mice subjected to CS after in vivo activation with interferon-gamma/lipopolysaccharide (CS + ACT), and in vitro infection with T. gondii tachyzoites. Peritoneal macrophages from CS + ACT mice showed decreased nitrite production compared to control but activated cells (ACT). Spleen cell proliferation to in vitro stimulation with the mitogens concanavalin A (Con A) and anti-CD3, and Toxoplasma lysate antigen (TLA) was measured in splenocytes obtained from BALB/c mice during the acute phase of infection with T. gondii. Mice subjected to CS and infection (CS + INF) had maximum splenocyte proliferation on days 8 and 15 followed by a subsequent decline on day 28 postinoculation (PI). In contrast, infected mice not subjected to stress (INF) showed decreased splenocyte proliferation on days 8 and 15 followed by an increase on day 28 PI. The rate of mortality was decreased in the CS + INF compared to the INF group during acute infection. These results suggest that CS may alter the pathogenesis of T. gondii infection by modulating acute-phase responses, provoking a state of transient disequilibrium between the host and parasite.     

Correlation between the avidity maturation of anti-HSP70 IgG autoantibody and recombination activating gene expressions in peripheral lymphoid tissues of Toxoplasma gondii-infected mice

The avidity maturation of anti-TgHSP70 IgG antibody produced by B-2 cells of BALB/c mice (a resistant strain) and that of anti-mHSP70 IgG autoantibody produced by B-1 cells of C57BL/6 mice (B6; a susceptible strain) was observed after Toxoplasma gondii infection. Recombination-activating genes (RAGs) were predominantly expressed in B-1 cells from peritoneal exudate cells (PECs) of T. gondii-infected B6 mice, while RAGs were expressed in B-2 cells from PECs of BALB/c mice. These results suggest that the involvement of RAG gene activations in the peripheral lymphoid tissues in the avidity maturation of anti-TgHSP70 IgG antibody and anti-mHSP70 IgG autoantibody in T. gondii-infected mice.     

Influence of low-density lipoprotein (LDL) receptor on lipid composition, inflammation and parasitism during Toxoplasma gondii infection

Intracellular replication of Toxoplasma gondii requires cholesterol uptake by host cell low-density lipoprotein receptor (LDLr), a critical element in atherosclerosis. We evaluated host parasitism, inflammatory responses and development of atherosclerosis in LDLr knockout (LDLr(-/-)) and their controls C57BL/6 mice infected with T. gondii. Our results show that T. gondii cysts were reduced in LDLr(-/-) mice when compared to C57BL/6 mice. However, in presence of hypercholesterolemic diet, parasite growth in LDLr(-/-) mice was similar to that seen in infected C57BL/6 mice. In presence of a hypercholesterolemic diet, T. gondii infection leads to a 60% reduction of serum triacylglycerol, total and atherogenic lipoprotein cholesterol. When aortic valve lesion was analyzed, infected mice showed a reduction of atherosclerotic lesion area as well as CD36 expression. MCP-1, SRA-I, SRA-II, ICAM-1 and VCAM-1 mRNA expression was kept similar between infected and control groups. Thus, despite the intense inflammatory process, the drastic reduction in serum lipids seems to limit the development of atherosclerosis in LDLr(-/-) mice infected with T. gondii. In conclusion, our results indicate that T. gondii employs host LDLr to acquire cholesterol and favor its growth. However, in the presence of hypercholesterolemia, T. gondii parasites are able to acquire cholesterol-rich lipoproteins through an alternative host receptor, and overcome LDLr deficiency, favoring host parasitism and impairing lipid loading of foam cells.     

In vivo recombinant interleukin 2 administration enhances survival against a lethal challenge with Toxoplasma gondii

Administration of recombinant interleukin 2 (rIL 2) resulted in a significant (p less than 0.01) decrease in mortality in mice infected with a dose of Toxoplasma gondii that killed 100% of untreated mice. Mice treated with rIL 2 had a significantly (less than 0.005) lower numbers of cysts in the brains. The protection afforded by rIL 2 could not be correlated with increased antibody synthesis or be explained by increased macrophage killing in the treated mice. Mice treated with rIL 2 after Toxoplasma infection demonstrated increased natural killer (NK) cell activity compared with either Toxoplasma-infected or rIL 2-treated mice. rIL 2 failed to reverse the suppressed proliferative response of lymphocytes to concanavalin A and lipopolysaccharide in mice acutely infected with a virulent strain of T. gondii. These results reveal that rIL 2 may have a remarkably protective effect against intracellular parasites.     

Mechanisms of killing of measles virus-infected cells by human lymphocytes: interferon associated and unassociated cell-mediated cytotoxicity

The recent interest in natural killer (NK) cells in immunosurveillance and the ability of infection with certain organisms to modulate NK activity led us to examine the influence of Toxoplasma gondii infection on mouse NK cells. Infection of BALB/c mice with 5 × 10³ virulent Toxoplasma intraperitoneally (ip) resulted in significantly enhanced NK activity in peritoneal exudate cells (PC) and in spleen cells (SC). Intravenous (iv) and subcutaneous (sc) challenge of BALB/c mice with Toxoplasma also resulted in enhanced natural killer (NK) activity in PC and SC. In BALB/c mice, as well as in other strains (A/J, C57BL/6, C3H/HeJ, CeH/HeN, [A/J × C3H]F₁), peak augmentation of PC and SC NK activity was observed 3 days following ip Toxoplasma challenge. Administration of silica to mice abolished Toxoplasma-induced NK cytotoxicity. BALB/c mice chronically infected with Toxoplasma had significantly higher endogenous NK activity than did controls in PC but not in SC. Chronically infected BALB/c mice boosted with virulent Toxoplasma ip exhibited significantly enhanced NK activity in PC but not in SC. Thus, acute and chronic infection with Toxoplasma modulates NK activity in addition to macrophage activation and thereby provides a system that should facilitate study of the relative contribution of NK cells and activated macrophages in resistance to tumor growth and spread.     

Toxoplasma gondii Hsp70 as a danger signal in toxoplasma gondii-infected mice

Toxoplasma gondii Hsp70, T gondii Hsp30/bag1, and surface antigen 1 messenger RNAs were shown to be useful in analyzing stage conversion of T gondii between bradyzoites and tachyzoites. The high-level expression of T gondii Hsp70 was correlated with mortality in interferon-gamma knockout mice infected with T gondii. Tgondii Hsp70 inhibited the induction of nitric oxide release by peritoneal macrophages of T gondii-infected mice. These findings identify T gondii Hsp70 as a danger signal during lethal, acute T gondii infection.     

First isolation of Neospora caninum from an aborted bovine fetus in Spain

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.     

A role for coccidian cGMP-dependent protein kinase in motility and invasion

The coccidian parasite cGMP-dependent protein kinase is the primary target of a novel coccidiostat, the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl] pyridine (compound 1), which effectively controls the proliferation of Eimeria tenella and Toxoplasma gondii parasites in animal models. The efficacy of compound 1 in parasite-specific metabolic assays of infected host cell monolayers is critically dependent on the timing of compound addition. Simultaneous addition of compound with extracellular E. tenella sporozoites or T. gondii tachyzoites inhibited [3H]-uracil uptake in a dose-dependent manner, while minimal efficacy was observed if compound addition was delayed, suggesting a block in host cell invasion. Immunofluorescence assays confirmed that compound 1 blocks the attachment of Eimeria sporozoites or Toxoplasma tachyzoites to host cells and inhibits parasite invasion and gliding motility. Compound 1 also inhibits the secretion of micronemal adhesins (E. tenella MIC1, MIC2 and T. gondii MIC2), an activity closely linked to invasion and motility in apicomplexan parasites. The inhibition of T. gondii MIC2 adhesin secretion by compound 1 was not reversed by treatment with calcium ionophores or by ethanol (a microneme secretagogue), suggesting a block downstream of calcium-dependent events commonly associated with the discharge of the microneme organelle in tachyzoites. Transgenic Toxoplasma strains expressing cGMP-dependent protein kinase mutant alleles that are refractory to compound 1 (including cGMP-dependent protein kinase knock-out lines complemented by such mutants) were used as tools to validate the potential role of cGMP-dependent protein kinase in invasion and motility. In these strains, parasite adhesin secretion, gliding motility, host cell attachment and invasion displayed a reduced sensitivity to compound 1. These data clearly demonstrate that cGMP-dependent protein kinase performs an important role in the host-parasite interaction.     

Isolation of Toxoplasma gondii from the keel-billed toucan (Ramphastos sulfuratus) from Costa Rica

Pectoral muscles from a captive keel-billed toucan (Ramphastos sulfuratus) from Costa Rica were fed to a Toxoplasma gondii-free cat, and the cat shed oocysts. Laboratory mice fed these oocysts developed antibodies to T. gondii in their sera and T. gondii tissue cysts in their brains. The DNA extracted from the brains of infected mice was characterized using 10 polymerase chain reaction-restricted fragment length polymorphic markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The isolate designated TgRsCrl was found to be non-clonal with Type I, II, and III alleles at different loci. This is the first isolation of T. gondii from this host.