Detection of Toxoplasma gondii by polymerase chain reaction in sera of acutely infected mice.

The presence of Toxoplasma gondii DNA was detected in sera of acutely infected mice by polymerase chain reaction. Adult mice were inoculated intraperitoneally with 5 x 10(3) T. gondii RH strain tachyzoites. Five mice were killed every 3 hr from 3 to 21 hr post infection (PI) and every day from 1 to 7 days PI. Toxoplasma gondii DNA was first detected in 60% of the infected mice 18 hr PI and in 100% of the animals 21 hr PI and from 1 to 7 days PI. No mice survived longer than 7 days.

Identification and initial characterization of Pneumocystis carinii soluble antigens in rabbit serum and lung lavage.

Lung lavage and serum samples from Azathioprin-treated (acute-phase infection) and untreated (non acute-phase infection) rabbits were used in the immunoblotting technique to look for Pneumocystis carinii (Pc) soluble antigens, using rabbit polyclonal antibodies raised against rabbit-derived Pc antigens and labeled with peroxidase. Analysis of the supernatant of lavage fluid after centrifugation to sediment intact organisms revealed components of approximately 80, 60-65, 55, 39 and 27 kDa in acute-phase samples. The components in the regions of 80, 60-65, 55 kDa and to a lesser extent 39 kDa were also present in non acute-phase lung lavage samples. In acute-phase serum samples, a major component of 80 kDa and minor components of about 65 and 39 kDa are detectable. The 80 and 65 kDa components are also detectable in some of the serum samples from the untreated rabbits. Immunofluorescent staining with FITC-conjugated affinity-purified antibodies to the 80, 60-65, 55, 39 or 27 kDa-components showed that they shared epitopes with both Pc cysts and trophozoites. The affinity-purified antibodies also cross-reacted in immunoblotting with several antigens in the Pc whole preparations. The putative Pc soluble antigens in serum and lung lavage were then isolated by affinity chromatography with polyclonal antibodies to Pc. Preliminary characterization of the column-extracted antigens revealed complete inactivation by trypsin whereas only the 55 and 80 kDa antigens bind to Concanavalin A. In conclusion, the results of this study suggest that Pc soluble antigens are present even in non acute-phase samples and only the low-molecular weight antigens (39 and 27 kDa) seem specific for the acute-phase. These findings are consistent with previous investigations reported by others that development of Pc could occur in nonimmu-nosuppressed rabbits.

Comparison of excretory/secretory and circulating antigens of Toxoplasma gondii by enzyme immunoassay and immunoblotting.

Toxoplasma gondii trophozoites (RH strain) were cultured in embryonic fibroblasts in order to study the kinetics of production of excretory/secretory antigens, and the results were compared to the production of circulating antigens in an in vivo mouse model. By capture-ELISA, excretory/secretory antigens were first detected on the fourth day of culture whereas circulating antigens were first detected 1 day after infection. Similar concentrations of antigens were detected in both models as evidenced by comparable absorbance values. By immunoblotting, the excretory/secretory antigens were also detected later compared to circulating antigens (day 4 vs day 1). Seven major polypeptides were detected in both antigen preparations, six of them having the same molecular mass (110, 75, 48, 30, 24 and 22 kDa).

Kinetic of circulating antigens by capture ELISA and immunoblotting in murine toxoplasmosis.

Capture ELISA and immunoblotting techniques were applied for the detection of Toxoplasma gondii (T.g) circulating antigens (CAG) in serum specimens of OF-1 mice infected with virulent RH, or cystogenic PGD strains. The capture ELISA assay allowed to detect CAG from the first day of infection to the death of animals with the two T.g strains, although CAG rates were higher when the RH strain was used. Immunoblotting confirmed these findings. Moreover, immunoblotting allowed to identify 7 CAG (MW: 22 kDa to 110 kDa) in serum specimens of mice infected with the RH strain and only 3 (MW: 75 kDa to 110 kDa) in serum specimens of mice infected with the PGD strain. These results are discussed in the light of the evolutive ways of experimental infection by T.g.

[Antigen analysis of Toxoplasma gondii using chromatography, polyacrylamide gel electrophoresis and immunoprecipitation]. / Analyse antigénique de Toxoplasma gondii par chromatographie, électrophorèse sur gel de polyacrylamide et immunoprécipitation.

Whole (EAT), cytoplasmic (EAC) and membranous (EAM) extracts were prepared from Toxoplasma gondii RH Strain trophozoites by means of osmotic lysis and/or ultra sonication. Sephacryl S 300 gel chromatography shows respectively 5, 4 and 2 protein fractions from EAT, EAC and EAM. Polyacrylamide gel electrophoresis (PAGE) allowed to detect 22 proteins (WM from 6 to 130 Kd) from cytoplasmic extract and 3 proteins (MW: 8, 30 and 65 Kd) from membranous extract. Immunoprecipitation techniques were performed with polyclonal antibodies against Toxoplasma prepared from serum and rabbits immunized with EAT. Counter electro-immunodiffusion (CEID) on cellulose acetate membrane was found to be more sensitive than double immunodiffusion and immunoelectrophoresis as it detected 8 lines with both cytoplasmic and membranous extracts. The latter seem to be composed of large numbers of proteins which are present in very low quantities difficult to detect in PAGE, but very antigenic and easily shown by CEID.

[Detection of circulating antigens of Toxoplasma gondii using immunoprecipitation reactions in murine toxoplasmosis]. / Détection des antigènes circulants de Toxoplasma gondii par réactions d'immunoprécipitation dans la toxoplasmose murine.

Circulating antigens (AGC) of Toxoplasma gondii were detected by counter electro-immunodiffusion (CEID) from the fourth day, in mice OF1 infected with 1,000 and 5,000 Toxoplasma gondii RH strain trophozoites. The mean number of precipitant lines increased from 1.6 on the fourth day to 5.8 on the seventh day, after which no animals survived. With the "Prugniaud" cystogenic strain no AGC were detected in mice inoculated with 100 and 200 cysts. Other techniques used, such as double immunodiffusion and immunoelectrophoresis, were shown to be less sensitive than CEID, and of little use for the detection of AGC. These AGC present antigenic similarities with some cytoplasmic fractions, particularly the FC3 fraction, and some membranous fractions such as FM1, separated by Sephacryl S300 gel chromatography.

[Job's syndrome--a case report (author's transl)].

A 4-year old boy with chronic eczema since early infancy had been admitted thrice to the hospital because of recurrent staphylococcal infections including furunculosis, lymphadenitis, pneumonia and pyothorax. Exhaustive immunologic studies revealed normal humoral, cell-mediated and non-specific immunities except extremely high serum IgE level and dysfunctions of granulocytes including decreased Fc and complement receptors, lowered chemotactic response (around the lower range of normal), slightly impaired intracellular killing of staphylococci and possibly impaired reactions to candida and BCG. In spite of long-term antimicrobial prophylaxis, cold abscesses continued to appear and the eczematous skin lesions waxed and waned.