A complement fixing toxoplasma antigen prepared from duck eggs and enhanced by phenol

Summary A method for the preparation of Toxoplasma antigen from infected duck chorioallantois is described. The reactivity of toxoplasma antigens can be enhanced by adding phenol. Department of bacteriology and serology.     

Verification of tween-ether antigen for complement fixation test in the diagnostics of toxoplasmosis

Tween-ether toxoplasma antigen for the complement fixation test was verified on a more extensive clinical material. From a series of 949 patient's sera, positive reaction was obtained in 44% of samples with the tween-ether antigen and in only 33.5% of samples with the FT antigen. All sera, giving positive results only with the tween-ether antigen, were also positive in the Sabin-Feldman test. The authors believe that by using this type of more sensitive antigen, containing also the cell-wall components of Toxoplasma gondii, it would be possible to standardize serological examination by the complement fixation test on the basis of an international standard serum.     

Serological survey of feline leukemia virus infection and the outcome of antibody-positive cats

A serological survey was carried out to examine the presence of antibodies against feline leukemia virus (FeLV) and feline oncornavirus-associated cell membrane antigen (FOCMA) in 208 cat sera collected at Teikyo University School of Medicine. Seven cats (3.4%) were positive for FeLV antibodies by enzyme-linked immunosorbent assay whereas no cat was positive for FOCMA antibody by indirect membrane immunofluorescent test. Anemia, leukemia and/or lymphoma formation were not observed in these FeLV antibody-positive cats. But among these seven cats, three were positive for toxoplasma antibodies. One of them was also positive for Chlamydia psittaci antibody and it died in pneumonia. Among the four toxoplasma antibody negative cats, one was died in eosinophilic granuloma. Furthermore, two of three cats, which were used for experiments, had cold and took therapy.     

Protection against toxoplasmosis in mice immunized with different antigens of Toxoplasma gondii incorporated into liposomes.

Different toxoplasma antigens were entrapped within liposomes and evaluated, in this form, for their ability to protect Swiss mice against toxoplasma infection: soluble tachyzoite antigen (L/TAg), tissue cyst (L/CAg), tachyzoite plus tissue cyst (L/TCAg) or purified antigen of tachyzoite (L/pTAg). The protein used in L/pTAg was purified from tachyzoites using a stage-specific monoclonal antibody which reacted at a molecular weight of 32 kD in SDS PAGE and silver stain using reduced condition. To compare the immuno-adjuvant action of liposomes and of Freund's Complete Adjuvant (FCA), another group of mice was immunized with soluble tachyzoite antigen (STAg) emulsified in FCA (FCA/TAg). Control groups were inoculated with (STAg) alone, phosphate-buffered saline (PBS), FCA with PBS (FCA/PBS) and empty liposomes (L/PBS). Mice were inoculated subcutaneously with these antigens six, four and two weeks before a challenge with 80 tissue cysts of the P strain of Toxoplasma gondii orally. All mice immunized with or without adjuvant showed a humoral response, as measured by Elisa. However, no correlation was found between antibody titer and protection against the challenge. All mice immunized with L/pTAg or L/TCAg survived (100), whereas 80% and 90% of mice from groups which received respectively PBS or FCA/PBS and L/PBS died. All mice immunized with antigens entrapped within liposomes (L/TAg, L/CAg, L/TCAg and L/pTAg) showed low numbers of intracerebral cysts.     

Plate Hemolysin Test for the Rapid Screening of Toxoplasma Antibodies

A plate hemolysin test was developed to screen serum specimens for the presence of toxoplasma antibodies. When we tested 130 sera by both this test and the standard toxoplasma dye test, we found the plate hemolysin test to be a rapid, sensitive, and economical method for detecting toxoplasma antibodies. In all but one instance it paralleled the dye test. A comparison of the results of testing six sera by the hemolysin, hemagglutination, and dye-test techniques suggested that the hemolytic antibodies were more closely related to hemagglutinating antibodies than to dye-test antibodies. We could not store sheep erythrocytes sensitized with toxoplasma lysate for more than 3 days without altering the sensitivity of the test. Concanavalin A proved to be an effective coupling agent for binding toxoplasma antigens to red-cell membranes, a quality attributed to its affinity for specific polysaccharide-combining sites.     

Toxoplasma gondii: a new diagnostic approach based on the specific binding of erythrocytes to lymphoid cells.

Red blood cells coated with toxoplasma antigen were bound to surface receptors, possibly of Ig nature, on lymphoid cells which appeared in the spleen of albino rats a few days after intraperitoneal injection of living T. gondii of the RH strain. Antigen-binding cells appeared before specific antibody, then declined in number, while the antibody response was progressing.Available information stresses the identity of antigen-binding cells with precursors of antibody-forming cells, thus pointing to the early occurrence of the immunocytoadherence phenomenon in the immune response. The applicability of these concepts to the field of human toxoplasmosis was proved by the demonstration of circulating lymphocytes specifically binding toxoplasma antigen early in the course of human toxoplasmic lymphadenitis and only in relation to active disease, thus providing an interesting approach to the early diagnosis and the detection of the active state of the disease.Possible clinical implications are discussed.     

Host response profile of human brain proteome in toxoplasma encephalitis co-infected with HIV

Background

Toxoplasma encephalitis is caused by the opportunistic protozoan parasite Toxoplasma gondii. Primary infection with T. gondii in immunocompetent individuals remains largely asymptomatic. In contrast, in immunocompromised individuals, reactivation of the parasite results in severe complications and mortality. Molecular changes at the protein level in the host central nervous system and proteins associated with pathogenesis of toxoplasma encephalitis are largely unexplored. We used a global quantitative proteomic strategy to identify differentially regulated proteins and affected molecular networks in the human host during T. gondii infection with HIV co-infection.

Results

We identified 3,496 proteins out of which 607 proteins were differentially expressed (≥1.5-fold) when frontal lobe of the brain from patients diagnosed with toxoplasma encephalitis was compared to control brain tissues. We validated differential expression of 3 proteins through immunohistochemistry, which was confirmed to be consistent with mass spectrometry analysis. Pathway analysis of differentially expressed proteins indicated deregulation of several pathways involved in antigen processing, immune response, neuronal growth, neurotransmitter transport and energy metabolism.

Conclusions

Global quantitative proteomic approach adopted in this study generated a comparative proteome profile of brain tissues from toxoplasma encephalitis patients co-infected with HIV. Differentially expressed proteins include previously reported and several new proteins in the context of T. gondii and HIV infection, which can be further investigated. Molecular pathways identified to be associated with the disease should enhance our understanding of pathogenesis in toxoplasma encephalitis.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-39) contains supplementary material, which is available to authorized users.     

High-level expression and purification of immunogenic recombinant SAG1 (P30) of Toxoplasma gondii in Escherichia coli

Surface antigen 1 (SAG1) of Toxoplasma gondii is a good candidate for diagnosis and vaccine development, but recombinant SAG1 produced in Escheichia coli often loses its specific immunogenicity due to the incorrect folding. In the present study, a truncated SAG1 was highly expressed in E. coli as a fusion protein, about 30% of the total protein of the cell lysate. After a simple purification and refolding procedure, purified rSAG1 can be recognized by human Toxoplasma-infective serum, and ELISA kits constructed by rSAG1 can sensitively and specifically detect toxoplasma infection.     

Technique for the detection of Toxoplasma gondii antigens in mouse urine

A simple and rapid staphylococcal coagglutination test for the detection of Toxoplasma gondii antigens in mice urine is described. A suspension of protein-A containing Staphylococcus aureus coated with rabbit hyperimmune serum was used as reagent. The sensitivity of the antigen assay was found to be at least 118 ng of the antigen protein per ml. No coagglutination was observed when the reagent was challenged against antigenic solutions of other parasites. The suitability of the method for detecting antigens of T. gondii in urine samples was studied by experimental toxoplasma infection in mice. Before the staphylococcal test, the urine samples were double serially diluted in 0.1 M PBS. From the second day on all samples from infected mice were positive at 1/16 dilution. At this dilution, all samples from non infected mice were negative or did not produce coagglutination. This method might be used in the rapid etiological diagnosis also in human cases of acute toxoplasmosis.     

The involvement of immunoproteasomes in induction of MHC class I-restricted immunity targeting Toxoplasma SAG1

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells and was exploited in the development of a DNA vaccine against the intracellular protozoan Toxoplasma gondii by constructing a chimeric DNA encoding a fusion protein between murine ubiquitin and the toxoplasma antigen SAG1. The SAG1 peptide was promptly degraded in antigen-presenting cells (APCs) transfected with the chimeric DNA. Degradation, however, was hampered by incubating the APCs with the proteasome inhibitor epoxomicin. Mice vaccinated with the DNA acquired potent protective immunity mediated by MHC class I-restricted CD8+ T cells against infection by the highly virulent Toxoplasma. The accelerated degradation and induction of immunity were dependent on the UPS since mice lacking an immuno-subunit of 20S proteasome, LMP7, lost these functions, although they were independent of the proteasome regulator PA28alpha/beta complex.     

The Prevalence of Toxoplasma Antibodies in Swine Sera in Finland

A total of 1847 swine sera obtained from the 10 largest abattoirs slaughtering swine in Finland were examined by ELISA for toxoplasma antibodies. The sample represented 0.64 % of the total number of swine slaughtered in these abattoirs over a period of 2 months. The prevalence of toxoplasma antibodies in swine sera was 2.5 %.     

Serologic test for antibody to Sarcocystis in cattle.

Soluble antigen was prepared from Sarcocystis zoites obtained from heart muscle of a bovine inoculated with sporocysts from canine feces and killed 120 days after infection. The antigen was used in an indirect hemagglutination (IHA) test and an agar gel diffusion test to detect antibody to Sarcocystis in experimentally infected cattle. IHA serum titers began to rise 30 to 45 days after infection and reached levels up to 1:39,000 90 days after infection. Sera collected under field conditions from 21 dairy cows had IHA titers ranging from 1:54 to 1:486. Since all cows appeared in good health, titers of 1:486 or less can probably be considered nonsignificant with regard to diagnosis of clinical disease. No positive Sarcocystis IHA titers were obtained with human sera previously found to be IHA positive for toxoplasma, indicating a lack of cross reactivity between antigens. Precipitins in the agar gel diffusion test appeared 30 days postinoculation and became very pronounced at 65 to 90 days.     

Incidence of acute symptomatic toxoplasma retinochoroiditis in south London according to country of birth.

OBJECTIVE--To determine the incidence of acute symptomatic toxoplasma retinochoroiditis presenting to ophthalmologists for patients born in Britain and elsewhere. DESIGN--Population based, cross sectional study. SETTING--11 districts in south Greater London. SUBJECTS--All patients presenting to NHS ophthalmologists with symptoms due to acute toxoplasma retinochoroiditis in 1992-3. MAIN OUTCOME MEASURE--Intraocular inflammation in association with a retinochoroidal scar, active adjoining retinitis, and IgG serum antibodies to toxoplasma. RESULTS--The estimated incidence of acute symptomatic retinochoroiditis for all people born in Britain was 0.4/100,000/year. If a mean of two symptomatic episodes per lifetime is assumed, 100 people born in Britain may be affected each year, about a fifth of the estimated 500-600 congenitally infected people born each year. CONCLUSIONS--A substantial proportion of people with acute symptomatic toxoplasma retinochoroiditis were born outside the country, and the number born in Britain was smaller than the number previously estimated to develop retinochoroidal lesions due to congenital toxoplasmosis. These findings suggest that prenatal screening for toxoplasmosis in Britain may be of limited benefit.     

Fetal immunization of baboons induces a fetal-specific antibody response

Neonates face a high risk of infection because of the immaturity of their immune systems. Although the transplacental transfer of maternal antibodies to the fetus may convey improved postnatal immunity, this transfer occurs late in gestation and may fail to prevent in utero infection. Both fetal immunization and in utero exposure to antigen can result in a state of immunologic tolerance in the neonate. Tolerance induction of fetal and premature infant lymphocytes has become a paradigm for neonatal responsiveness. However, fetal IgM responses have been demonstrated to maternal immunization with tetanus toxoid and to congenital infections such as rubella, toxoplasma, cytomegalovirus and human immunodeficiency virus. Moreover, 1-week-old infants can respond to standard pediatric vaccination, and neonates immunized with polysaccharide antigens do not develop immunologic tolerance. Here, direct immunization of the baboon fetus with recombinant hepatitis B surface antigen produced a specific fetal IgG antibody response. No specific maternal antibody response was detected, eliminating the possibility of vertical antibody transmission to the fetus. Some infants also responded to later vaccinations with hepatitis B surface antigen, indicating that no immunological tolerance was induced by prior fetal immunization. These results characterize the ability of the fetal immune system to respond to in utero vaccination. We demonstrate that active fetal immunization can serve as a safe and efficient vaccination strategy for the fetus and neonate.     

Toxoplasmal and Toxocaral Infections: A Clinical Investigation into Their Relationship

An investigation was carried out to determine whether a relationship existed between infections with Toxoplasma gondii and Toxocara canis. Antibodies were sought by the toxoplasma dye test and the toxocara skin sensitivity test. Sixty-seven patients were examined in the United Kingdom; 38 had positive toxoplasma dye tests, two of these being toxocara-positive; and 29 had negative dye tests, six of them being toxocara-positive. From the total of eight toxocarapositive patients antibodies to toxoplasma were detected in two, an incidence no greater than that expected for a normal population.Sera from 60 toxocara-positive African patients were examined; 20 possessed antibodies to Toxoplasma. Analysis according to age group or geographical location indicated that this incidence was no greater than that expected for patients without evidence of toxocaral infection.This study therefore showed no causal or clinically important relationship between toxoplasmal and toxocaral infections.     

Toxoplasma gondii: trypsin treatment of trophozoites for enhanced immune cytolysis in the dye test

The Sabin-Feldman dye test for the detection of toxoplasma antibodies has been modified by the use of fresh guinea pig serum and of trypsinized toxoplasma trophozoites. Guinea pig serum at a final concentration as low as 1.0–1.5% has been found sufficient to give satisfactory results. The use of trypsinized parasites has been observed to enhance the sensitivity of the titer. It is suggested that the organisms are coated with a trypsin labile substance which may be responsible for preventing access of antibodies to the parasite cytoplasm. The complement system in the serum may be the only essential accessory factor in this respect.     

Toxoplasma gondii: Microassay to differentiate toxoplasma inhibiting factor and interleukin 2

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [³H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin -and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.     

Cytological study of a continuous swine embryonic kidney cell line with a persistent Toxoplasma infection]

Invasion of the PEK cell culture by toxoplasma did not cause any sharp disruption of the vital activity of the cells and failed to lead to their rapid degeneration. The observed changes in the morphology of the host cells were apparently chiefly the result of a purely mechanical pressure of the vacuoles on the nucleus and the cytoplasm, although the extreme forms of disturbances of the cell morphology were possibly associated with their greater injury. No complete "selfpurification" of the cells apparently occurred, since despite the marked reduction in the number of parasites a possibility of reinfection with toxoplasma of the culture at the later periods after the infection could not be excluded.     

Determination of specific immunoglobin G avidity in serodiagnosis of toxoplasmosis

A total of 37 serum samples from pregnant women were examined for the avidity of toxoplasma IgG antibodies using commercially available enzyme immunoassay (Vidas Toxo IgG Avidity, bio Merieux). Low values of the avidity index were found for 4 samples and borderline ones were found in further 3 cases. Out of 19 serum samples showing the presence of IgG and IgM toxoplasma antibodies, 13 had high avidity index, including 9 samples from women in the first trimester of pregnancy. The results of examinations in these 9 cases allowed to exclude the possibility that toxoplasma infection was acquired during gestation. It is to be concluded that determinations of antitoxoplasma IgG avidity should become routine in every pregnant woman positive in the test for specific IgM antibody.     

Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique

Proteins of Toxoplasma gondii were separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. Immunologically reactive polypeptides were detected by human sera with previously known toxoplasma antibody levels. Heavy chain-specific, peroxidase-conjugated anti-human immunoglobulins were used as the indicator antibodies for the separate identification of IgG and IgM reactive polypeptides. IgG toxoplasma antibodies reacted with several antigens of M_r ≈27 000–67 000, while toxoplasma-specific IgM seemed to detect only a few polypeptides. The M_r of 35 000 for the dominating IgM reactive polypeptide was observed.