Toxoplasma gondii: Electron microscopic study on the dye test reaction

When intact Toxoplasma trophozoites were stained with isotonic alkaline methylene blue solution, the organelles rich in nucleic acid, i.e., nucleus, free and membrane-bound ribosomes appeared as electron dense areas. When the parasites were incubated with the anti-Toxoplasma antibody and the accessory factor, swelling of the surface membrane occurred first, followed by destruction of the inner structures. In the dye test positive parasites, there were no definite organelles recognizable, as there were in the intact parasites. By the negative staining method, holes (defects) with dark central portions were observed on the surface of the parasites treated with the antibody and the accessory factor, the diameter of the holes measuring about 10–11 nm. These holes, which tended to occur in clusters, were each surrounded by a clear ring.     

Toxoplasma gondii: purification of trophozoites propagated in cell culture.

A new procedure is described for the purification of trophozoites from the virulent RH strain of Toxoplasma gondii propagated in baby hamster kidney (BHK-21) cell cultures. The culture medium containing host cell debris and trophozoites was filtered through glass-wool filtering fiber, which removed most host cell material. The filtrate containing trophozoites was centrifuged, and the trophozoite pellet was resuspended and washed in phosphate-buffered saline. An average of about 75% of the original number of trophozoites was recovered. No loss of trophozoite viability was observed as determined by the rate of host cell culture monolayer destruction. The amount of host cell material contamination in the final trophozoite fraction was negligible as determined by measuring radioactivity in the trophozoite fraction after cofiltration with noninfected host cell material which had been prelabeled with radioactive precursors.     

Schistosoma mansoni: The role of the complement C3-activating system in the cercaricidal action of normal serum

Living Schistosoma mansoni cercariae incubated with normal serum from several species were damaged, as revealed by immobilization of their tails, uptake of methylene blue dye and the incapacity to infect appropriate vertebrate hosts. The following data indicate that the cercaricidal action of normal sera is dependent on the complement system, activation of the system proceeding through the alternate (properdin) pathway: (a) consumption of appreciable amounts of total hemolytic complement during the incubation of fresh serum with living cercariae; (b) the preferential consumption of the late reacting components and except for human C₄, only limited consumption of the classical early components; (c) the inactivity of sera depleted of C₃ or properdin; (d) the selective requirement for the presence of Mg²⁺ in the incubation mixture; (e) the full cercaricidal effect of C₄—deficient guinea pig serum; and (f) the conversion of C₃ after incubation of normal serum with the cercariae to electrophoretically faster migrating products. Immunofluorescence analyses indicated that the structures responsible for the complement activation are present in the cercarial coat.     

Identification of tubular heparan sulfate as a docking platform for the alternative complement component properdin in proteinuric renal disease

Properdin binds to proximal tubular epithelial cells (PTEC) and activates the complement system via the alternative pathway in vitro. Cellular ligands for properdin in the kidney have not yet been identified. Because properdin interacts with solid-phase heparin, we investigated whether heparan sulfate proteoglycans (HSPG) could be the physiological ligands of properdin. Kidneys from proteinuric rats showed colocalization of syndecan-1, a major epithelial HSPG, and properdin in the apical membranes of PTEC, which was not seen in control renal tissue. In vitro, PTEC did not constitutively express properdin. However, exogenous properdin binds to these cells in a dose-dependent fashion. Properdin binding was prevented by heparitinase pretreatment of the cells and was dose-dependently inhibited by exogenous heparin. ELISA and surface plasmon resonance spectroscopy (BIAcore) showed a strong dose-dependent interaction between heparan sulfate (HS) and properdin (K(d) = 128 nm). Pretreatment of HSPG with heparitinase abolished this interaction in ELISA. Competition assays, using a library of HS-like polysaccharides, showed that sulfation pattern, chain length, and backbone composition determine the interaction of properdin with glycosaminoglycans. Interestingly, two nonanticoagulant heparin derivatives inhibited properdin-HS interaction in ELISA and BIAcore. Incubation of PTEC with human serum as complement source led to complement activation and deposition of C3 on the cells. This C3 deposition is dependent on the binding of properdin to HS as shown by heparitinase pretreatment of the cells. Our data identify tubular HS as a novel docking platform for alternative pathway activation via properdin, which might play a role in proteinuric renal damage. Our study also suggests nonanticoagulant heparinoids may provide renoprotection in complement-dependent renal diseases.     

Toxoplasma gondii: disease patterns in mice treated with the folate antagonist methotrexate.

Methotrexate (MTX), a folic acid antagonist, was administered to Toxoplasma-infected mice in an attempt to inhibit acquired resistance to the parasite. Several time- and dose-dependent drug regimes were examined, with the following results.MTX administered during the first week of infection converted subclinical, nonlethal infections into severe disease with pronounced morbidity, either with or without high mortality. Depending on the drug regime employed, three different patterns of disease emerged. Constant findings in the MTX-treated mice were persistence of Toxoplasma trophozoites in peritoneal exudate and viscera; earlier appearance and increased numbers of cysts in the brain; development of many cysts in large, grapelike clusters; and a severe, disseminated meningoencephalitis.When administration of MTX was delayed until the twelfth day postexposure, its infection-modifying ability was lost, indicating that immunogenesis by this time has provided a high level of acquired resistance to Toxoplasma.MTX had no discernible effects when started 30 days postexposure. Reactivation of the latent infection did not occur.     

Ascaris suum and Toxocara canis: Egg extract antigens in guinea pigs and the macrophage migration inhibition test

Guinea pigs immunized with 500 μg of either Ascaris suum or Toxocara canis egg extracts, emulsified with Freund's complete adjuvant, were skin tested with both the homologous and heterologous antigens. Cross-reactions were observed in both groups. Migration of macrophages from sensitized animals was more inhibited by homologous than by heterologous antigens. Lymph-node lymphocytes from sensitized animals were stimulated to incorporate [⁸H]thymidine similarly by both antigens.     

Toxoplasma gondii: comparison of a rhoptry-ELISA with IFAT and MAT for antibody detection in sera of experimentally infected pigs

Indirect ELISA and IFAT have been reported to be more sensitive and specific than agglutination tests. However, MAT is cheaper, easier than the others and does not need special equipment. The purpose of this study was to compare an enzyme linked immunosorbent assay using crude rhoptries of Toxoplasma gondii as coating wells (r-ELISA) with indirect fluorescence antibody test (IFAT) and modified agglutination test (MAT) to detect anti-T. gondii antibodies in sera of experimentally infected pigs. Ten mixed breed pigs between 6.5 and 7.5 weeks old were used. All pigs were negative for the presence of T. gondii antibodies by IFAT (titre < 16), r-ELISA (OD < 0.295) and MAT (titre < 16). Animals received 7x10(7) viable tachyzoites of the RH strain by intramuscular (IM) route at day 0. Serum samples were collected at days -6, 0, 7, 14, 21, 28, 35, 42, 50, and 57. IFAT detected anti-T. gondii antibodies earlier than r-ELISA and MAT. The average of antibody levels was higher at day 35 in IFAT (Log10=2.9) and in MAT (Log10 = 3.5), and at day 42 in r-ELISA (OD = 0.797). The antibody levels remained high through the 57th day after inoculation in MAT, and there was a decrease tendency in r-ELISA and IFAT. IFAT was used as "gold standard" and r-ELISA demonstrated a higher prevalence (73.3%), sensitivity (94.3%), negative predictive value (83.3%), and accuracy (95.6%) than MAT. Kappa agreements among tests were calculated, and the best results were shown by r-ELISAxIFAT (kappa = 0.88, p < 0.001). Cross-reaction with Sarcocystis miescheriana was investigated in r-ELISA and OD mean was 0.163 +/- 0.035 (n = 65). Additionally, none of the animals inoculated with Sarcocystis reacted positively in r-ELISA. Our results indicate that r-ELISA could be a good method for serological detection of T. gondii infection in pigs.     

The in vivo conformation of the plastid DNA of Toxoplasma gondii: implications for replication

The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.     

Investigation Into the Humaneness of Slaughter Methods for Guinea Pigs (Cavia porcelus) in the Andean Region

Guinea pigs (Cavia porcelus) are an important source of nonhuman animal protein in the Andean region of South America. Specific guidelines regarding the welfare of guinea pigs before and during slaughter have yet to be developed. This study critically assessed the humaneness of 4 different stunning/slaughter methods for guinea pigs: cervical neck dislocation (n = 60), electrical head-only stunning (n = 83), carbon dioxide (CO₂) stunning (n = 21), and penetrating captive bolt (n = 10). Following cervical neck dislocation, 97% of guinea pigs had at least 1 behavioral or cranial/spinal response. Six percent of guinea pigs were classified as mis-stunned after electrical stunning, and 1% were classified as mis-stunned after captive bolt. Increased respiratory effort was observed during CO₂ stunning. Apart from this finding, there were no other obvious behavioral responses that could be associated with suffering. Of the methods assessed, captive bolt was deemed the most humane, effective, and practical method of stunning guinea pigs. Cervical neck dislocation should not be recommended as a slaughter method for guinea pigs.     

Feasibility and optimization of wastewater treatment by chemically enhanced primary treatment (CEPT): a case study of Huangshi

Carbon and nutrients as well as suspended solids (SS) removal by chemically enhanced primary treatment (CEPT) were conducted in the Qingshan wastewater treatment plant in Huangshi, Hubei Province. Feasibility of this process for wastewater treatment were investigated in detail by comparing the removal performance of three inorganic chemical coagulants (polyaluminium chloride, polyaluminium ferric chloride [PAFC] and poly ferric sulfate) individual or couple with poly acrylamide, optimizing the conditions during CEPT by both single factor analysis and orthogonal test designs. The results of this study demonstrated that CEPT turned out to be an effective method for wastewater treatment, with PAFC as the optimal coagulant, which showed preeminent removal capacity for chemical oxygen demand, total phosphorus and SS. The optimal working condition could be at pH 7.0, settling time 15 min, and velocity gradient of 174.80 and 15.56 s^?1 for mixing and reaction phase respectively. While the coagulant dosage depends on raw water attributes, which had a decisive effect on CEPT treatment performances. However, the three coagulants behaved poorly in nitrogen removal.     

Infection of male rats with Toxoplasma gondii results in enhanced delay aversion and neural changes in the nucleus accumbens core

Rats infected with the protozoan parasite Toxoplasma gondii exhibit reduced avoidance of predator odours. This behavioural change is likely to increase transmission of the parasite from rats to cats. Here, we show that infection with T. gondii increases the propensity of the infected rats to make more impulsive choices, manifested as delay aversion in an intertemporal choice task. Concomitantly, T. gondii infection causes reduction in dopamine content and neuronal spine density of the nucleus accumbens core, but not of the nucleus accumbens shell. These results are consistent with a role of the nucleus accumbens dopaminergic system in mediation of choice impulsivity and goal-directed behaviours. Our observations suggest that T. gondii infection in rats causes a syndromic shift in related behavioural constructs of innate aversion and making foraging decisions.     

Inducible nitric oxide synthase in innate immune cells is important for restricting cyst formation of Toxoplasma gondii in the brain but not required for the protective immune process to remove the cysts

Significantly larger numbers of Toxoplasma gondii cysts were detected in the brains of RAG1^?/?NOS2^?/? than RAG1^?/? mice following infection. In contrast, the cyst numbers markedly decreased in a same manner in both strains of mice after receiving CD8⁺ immune T cells. Thus, NOS2-mediated innate immunity is important for inhibiting formation of cysts in the brain but not required for the T cell-initiated cyst removal, which is associated with phagocyte accumulation. Treatment with chloroquine, an inhibitor of endolysosomal acidification, partially but significantly inhibited the T cell-mediated cyst removal, suggesting that phagosome–lysosome fusion could be involved in the T. gondii cyst elimination.     

Polysaccharide conjugated laccase for the dye decolorization and reusability of effluent in textile industry

Nine different polysaccharides were screened for conjugation with laccase and evaluated for pH and thermal stability. All the polysaccharides decreased the thermal and pH stability of laccase at 50 °C and 60 °C, where conjugation with gum Arabic showing the most pronounced effect. Thermal instability of gum Arabic conjugated laccase was affirmed by differential scanning calorimeter while the structural changes in the conjugated laccase responsible for thermal instability was analysed by fluorescence spectrophotometer. The gum Arabic conjugated laccase showed an unusually high tolerance to sodium chloride, thermal instability and lower stability in alkaline conditions. Gum Arabic conjugated laccase was found to decolorize Remazol brilliant blue R in the textile effluent at a slower rate without any microbial growth which was unlike that observed in effluent treated with free laccase. Further, effluent treated with conjugated laccase enabled its reuse as liquor for the dyeing to get desired shade.     

Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.     

Evidence for the existence of a proteasome in Toxoplasma gondii: intracellular localization and specific peptidase activities.

The proteasome is a large intracellular protein complex whose main function is proteolytic removal of damaged proteins. It has recently been shown that the proteasome has a crucial role in the pathogenesis of protozoan parasites. We attempted to characterize the proteasome of T. gondii (RH strain). In immunoblot experiments, we showed that MCP231 monoclonal antibody, directed against the human 20S proteasome, labelled homologous proteins in T. gondii with a pattern similar to that observed in mammalian cells. The study of in vitro proteolytic activities showed that chymotrypsin-like activity (the only activity obtained with archaebacteria) was present in Toxoplasma, with Km and specific activity values close to those observed with eukaryotic cells. Immunofluorescence studies showed that the Toxoplasma proteasome predominated in the cytosol.     

A single in vivo-selected point mutation in the active center of Toxoplasma gondii ferredoxin-NADP+ reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin

Electron transfer between plant-type [2Fe-2S] ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) depends on the physical interaction between both proteins. We have applied a random mutagenesis approach with subsequent in vivo selection using the yeast two-hybrid system to obtain mutants of Toxoplasma gondii FNR with higher affinity for Fd. One mutant showed a 10-fold enhanced binding using affinity chromatography on immobilized Fd. A single serine-to-arginine exchange in the active site was responsible for its increased affinity. The mutant reductase was also enzymatically inactive. Homology modeling of the mutant FNR-Fd complex predicts substantial alterations of protein-FAD interactions in the active site of the enzyme with subsequent structural changes. Collectively, for the first time a point mutation in this important class of enzymes is described which leads to greatly enhanced affinity for its protein ligand.     

Neospora caninum: detection in wild rabbits and investigation of co-infection with Toxoplasma gondii by PCR analysis

Neospora caninum is an important pathogen of cattle causing significant economic loss. There is much current interest in wild animal reservoirs for this parasite. The role of the rabbit in this is currently unknown. DNA samples from the brains of wild rabbits (Oryctolagus cuniculus) collected from the Malham area of the Yorkshire dales were investigated by species-specific PCR for the presence of N. caninum and Toxoplasma gondii. We found prevalences of N. caninum of 10.5% (6/57) and T. gondii of 68.4% (39/57) with 8.8% (5/57) co-infected. Strain typing of T. gondii positive rabbits revealed strain types I-III were present in this population. Investigation of tissue distribution determined N. caninum DNA was most often detected in the brain and heart, less often in the tongue and not in the liver. To our knowledge this is the first report of N. caninum detection in naturally infected wild rabbits.