Arrays of repetitive DNA elements in the largest chromosomes of Toxoplasma gondii.

A novel tandemly repeated DNA structure of Toxoplasma gondii that meets the requirements assigned for satellital DNA was characterized. A DNA fragment of 1002 bp contains two different elements of repetitive DNA families named ABGTg7 and ABGTg8.2. Both repeats are members of a more complex tandem structure where ABGTg7-like monomers can be arranged either as direct tandems or flanked by other related or non-related repeats. Pulse-field gel electrophoresis analysis showed that these repeats hybridize with the largest T. gondii chromosomes. Bal31 sensitivity assays indicated that these elements are located near the telomeres and along other regions too. Five genomic lambda phages were isolated and two different completed clusters of the repeated structure were analyzed.

Detection of human Toxoplasma-specific immunoglobulins A, M, and G with a recombinant Toxoplasma gondii rop2 protein.

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2196-561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2196-561 as the antigen substrate. The analyzed sera were divided according to T. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG+ IgA- IgM-; n = 35), group B (IgG+ IgA+ IgM+; n = 21), group C (IgG+ IgA+ IgM-; n = 5), and group D (IgG+ IgA- IgM+; n = 16). Twenty-six T. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2196-561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2196-561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.

Screening for active toxoplasmosis in patients by DNA hybridization with the ABGTg7 probe in blood samples.

We report the potential use of a specific Toxoplasma gondii DNA probe (ABGTg7). We applied a dot blot hybridization assay to blood samples for the diagnosis of cerebral toxoplasmosis (CT), acute toxoplasmic lymphadenopathy (ATL), and disseminated toxoplasmosis in transplant recipients (TRs). We studied a total of 84 individuals: 38 patients and 46 controls. We found positive hybridization signals for 12 (66.7%) of 18 patients with confirmed CT, 9 (52.9%) of 17 patients with ATL, and 2 (66.7%) of 3 TRs. PCR assays were performed in parallel for patients with ATL, resulting in T. gondii DNA detection for 10 patients (58.8%). A comparative study between dot blot and PCR assays performed with the blood of mice that had been experimentally infected with tachyzoites gave similar results: 60 and 70% positive results, respectively. Finally, the sum of positive values obtained by both DNA tests (dot blot assay plus PCR) increased the rate of positivity for ATL patients to 76.4%. These results demonstrate that the T. gondii ABGTg7 repetitive DNA element is an additional useful resource for diagnosing Toxoplasma parasitemia in patients with CT and ATL and in TRs. Thus, our ABGTg7-based dot blot test may lead to an improvement in T. gondii detection methods in patients with acute toxoplasmosis.

Use of a monoclonal antibody and a DNA probe for diagnosing acute toxoplasmosis in ambiguous cases.

An unusual case of cerebral toxoplasmosis in an HIV negative 11 year old child is reported. Central nervous system disease was assessed immunohistochemically in a brain biopsy specimen with TgP8--a specific monoclonal antibody against Toxoplasma gondii antigens--thus confirming IgG and IgM serology, technetium scan findings, and clinical data. In addition, an active parasitaemia was confirmed by DNA in situ hybridisation assay in white cells using an ABGTg4 probe. The child recovered after specific T gondii treatment. Follow up six months later showed that he was immunodeficient.

Deoxyribonuclease activity in Echinococcus granulosus protoscoleces and hydatid fluid.

Deoxyribonuclease activity was detected in E. granulosus protoscoleces from sheep hydatid cysts by electrophoresis in agarose gels of DNA fragments obtained after incubation of integral DNA with a protoscoleces preparation. Preliminary characterization showed that deoxyribonuclease activity was optimal at neutral-alkaline pH, magnesium ions were required, and it was able to digest different types of DNA, making random cuts. Electrophoresis in DNA-containing sodium dodecylsulfate (SDS)-polyacrylamide gels indicated a relative molecular mass, under non-reducing conditions, of 32 kDa. Deoxyribonuclease activity was also found in sheep hydatid fluid. It shared optimal pH, ionic and substrate requirements with the enzyme from protoscoleces but had a higher relative molecular mass (40 kDa), the same as that of normal sheep serum deoxyribonuclease.

A simple and economic slide micro-immunoenzymatic (micro-SIA) test for epidemiological studies of toxoplasmosis.

A slide micro-immunoenzymatic assay (micro-SIA) to detect antibodies to non-particulate Toxoplasma gondii antigens is described. This assay allows the diagnosis of toxoplasmosis infection in about 1 hr. Twenty-four determinations can be performed per slide. Five hundred ng of antigen and 5 or 10 microliters drop of each reactive are necessary per well. The clear contrast of colours obtained for negative and positive sera after the test is finished, allows direct discrimination of the results. However, it is possible to quantify the results of the reaction using a minireader. Sera dilution cutoff value, determined as the most frequent titre for the general population, is 1:100. The toxoplasma micro-SIA correlates well with indirect immunofluorescence (IIF), its sensitivity is at least three times as much as IIF. The test has an intra and inter assay variation coefficient of 5.46% and of 6.24% respectively. Sera obtained at random from argentinian people were analyzed and a 56% of infection was found. The main features of the Toxoplasma micro-SIA are its simplicity, sensitivity, reproducibility, and the virtual absence of background making it very suitable for screening tests.

Early diagnosis of toxoplasmic encephalitis in AIDS patients by dot blot hybridization analysis.

Central nervous system toxoplasmosis is a life-threatening infection with a mortality rate of higher than 60%. An early and rapid diagnosis is important for effective treatment of the disease. A new approach for detection of cerebral toxoplasmosis is described here. DNAs extracted from cells in cerebrospinal fluid samples (0.3 to 0.8 ml) of patients suspected of having cerebral toxoplasmosis were analyzed by a dot blot hybridization technique. A highly repetitive DNA sequence of Toxoplasma gondii (ABGTg4) was nonisotopically labelled with digoxigenin-dUTP and used as a specific DNA probe. Four of six patients analyzed gave positive signals in our hybridization assay. Two of them recovered with pyrimethamine-sulfadiazine, a drug recommended for treatment of toxoplasmosis. The other two patients with positive signals died soon after diagnosis. Patients with negative signals were found to suffer from mycobacterial infection (patient 1) or varicella-zoster virus infection (patient 6).

Cloning of repetitive DNA sequences from Toxoplasma gondii and their usefulness for parasite detection.

Genomic DNA of Toxoplasma gondii was digested with the restriction endonuclease Hpa II and the resulting repetitive DNA sequences were visualized after electrophoresis on agarose gels and staining with ethidium bromide. Three repetitive DNA sequences were isolated and cloned in the plasmid pUC19. The recombinant plasmids (pTg8, pTg4 and pTg1) had inserts of 840, 440, and 180 basepairs, respectively. The estimated copy number of these cloned sequences in the T. gondii genome was approximately 800-1,000 for pTg4, 150-200 for pTg8, and 30-40 for pTg1. In dot-blot hybridization tests, pTg4 was able to detect as little as 80 pg of purified T. gondii DNA or 1,000 parasites in the presence or absence of 1.5 x 10(6) human or mouse leukocytes. No cross-hybridization was detected with 10 micrograms of either human and mouse DNA or 100 ng of DNA from other parasites (Eimeria tenella, E. acervularia, Trypanosoma cruzi, and Leishmania donovani), or among the three DNA probes. Each probe identified T. gondii tachyzoites in tissue (liver and spleen) obtained from experimentally infected mice in which histologic damage was detected. In addition, early detection of T. gondii in brain tissue and blood samples was possible with the pTg4 probe.

Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites. Preliminary characterization of its genome.

A rapid and simple technique for the purification of Toxoplasma gondii tachyzoites was developed. Highly purified parasites were obtained from the peritoneal exudates of infected mice by means of two consecutive discontinuous sucrose gradients run at low speed (10,000xg, 30 min). Parasites obtained by this method conserved its biological activity. Hybridization studies with DNA from healthy mice and from purified tachyzoites preparations demonstrated that Toxoplasma gondii tachyzoites DNA could be obtained with better than 90% purity. Preliminary studies with DNA endonucleases showed the presence in the tachyzoites genome of highly repetitive sequences.

Evaluation of neutrophil activity and circulating immune complexes levels in diabetic patients.

We have studied chemiluminescence produced by neutrophils stimulated by opsonized zymosan in insulin dependent (IDD) and non insulin dependent (NIDD) diabetic patients. Chemiluminescence was evaluated as the integral and maximum peak, total time and time to maximum peak of the response curve to opsonized zymosan. These values were then compared with circulating immune complexes (CIC) and glucose levels. Both IDD and NIDD patients had significantly higher values of chemiluminescence and CIC than normal controls. We also observed that patients who had the highest values of CIC and chemiluminescence levels were the ones with clinical microvascular complications.

Extensive deletions in the Q region of the mouse major histocompatibility complex.

By use of Southern blot analyses and low copy number probes, the fine structure of the Q region of the mouse major histocompatibility complex was studied in more detail. With a probe recognizing the even-numbered genes Q4, Q6, and Q8, it was evident that Q4 and/or the regions flanking Q4 are polymorphic, whereas Q6 and Q8, and their flanking regions are nonpolymorphic. Perhaps the most noteworthy finding is that at least two strain haplotypes, H-2k and H-2f, possessed extensive deletions in the Q region. The most striking deletion was found in the H-2f haplotype, where the Q1 through Q9 genes appear to be missing. Because of these extensive deletions the functional importance of the Q region is questioned.

Antigenic mosaic of Methanosarcinaceae: partial characterization of Methanosarcina barkeri 227 surface antigens by monoclonal antibodies.

Hybridomas were constructed with spleen cells from mice immunized against Methanosarcina barkeri 227. The reaction with the resulting monoclonal antibodies identified two antigenic determinants. Determinant 8A is present in M. barkeri 227, where it is accessible to antibody on whole bacterial cells. 8A is undetectable in (or absent from) M. barkeri R1M3, an immunologically closely related strain. Determinant 8C is present in both strains, but with M. barkeri 227 it is found only in extracts and cannot be demonstrated in whole cells. It therefore appears to be hidden. A soluble form of antigen 8A (antigen 227) was obtained treating whole M. barkeri 227 cells with absolute methanol. This antigen was further purified by affinity chromatography with antibody 8A. Chemical and immunochemical analyses of these preparations showed that antigen 227 is a high-molecular-weight (4 X 10(5)) structure composed mainly of one carbohydrate, glucose, and small amounts of amino acids. Its solubility properties suggest that this molecule is associated with a lipid moiety.

Circulating immune complexes and B lymphocytes in diabetic patients with and without microangiopathy.

Circulating immune complexes (CIC) were evaluated in 57 diabetic patients: 28 were insulin-dependent (IDD) and 29 were non insulin-dependent (NIDD) subdivided according to the presence or absence of microangiopathy. The following techniques were used: 1) binding to human red blood cells through C3b complement fraction and posterior radioimmunoassay with 125I labeled anti human IgG (HRBC RIA test); and 2) CIC precipitation with 3.5% polyethylenglycol (PEG test). The percentage of circulating B lymphocytes was evaluated simultaneously in the same patients, using a) direct immunofluorescence techniques (surface IgC cells) and b) cells with complement C3b fraction receptors (EAC rosettes). Twenty normal donors were studied simultaneously as controls. Our results showed that CIC levels were significantly higher in both groups of patients when compared to normal controls. Values for IDD and NIDD were 48.55 +/- 5.97 and 34.68 +/- 3.08 microgram/ml, respectively, for HRBC RIA test and 0.53 +/- 0.07 and 0.41 +/- 0.04 O.D., respectively, for PEG test, while control values were 26.63 +/- 2.12 microgram/ml for HRBC RIA test and 0.26 +/- 0.03 O.D. for PEG test. IDD patients with microangiopathy presented higher CIC levels as measured by HRBC RIA test than IDD without microvascular complications, while no difference was found within the NIDD group. An increase in the proportion of cells bearing surface IgG was observed in IDD and NIDD patients when compared to controls. By contrast, no difference was observed when EAC rosettes-forming cells were evaluated.

Carbohydrate composition of specific rat epididymal protein.

Previous results demonstrated that androgen-dependent rat specific epididymal proteins (SEP) were bound to spermatozoa during their maturation in the epididymis. This paper describes the purification of glycoprotein DE, which constitutes 40% of SEP, and the identification and semiquantitative determination of the sugars forming its oligosaccharide chain. Affinity chromatography on Sepharose-Concanavalin A produced a sample of D-E 95% pure in which 10.5 g of sugar were present per 100 g protein. The percentual composition of the oligosaccharide was D-mannose 19%; D-galactose 3%; N-acetyl-D-glucosamine 33%; N-acetyl-neuraminic acid 31% and D-glucose 13%.