Serodiagnosis of acute toxoplasmosis using a recombinant form of the dense granule antigen GRA6 in an enzyme-linked immunosorbent assay

We developed an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of acute toxoplasmosis that used the recombinant granule antigen GRA6-GST as diagnostic antigen for the detection of IgG antibodies to Toxoplasma gondii in human sera. A total of 431 sera obtained from 336 patients with acute and chronic toxoplasmosis and from patients who were not infected with T. gondii were tested. Sera from patients with acute T. gondii infection, chronic infection, and no infection showed different absorbance values. For discrimination between the presence and the absence of acute toxoplasmosis the assay reached a specificity of 99.6%. Only one of the sera without significant anti-T. gondii. IgM antibodies showed a positive reaction to rGRA6-GST. The assay showed good intra- and interassay reproducibility (CV 6%/14%). We included a glutathione S-transferase (GST)-IgG enzyme immunoassay as a control assay in this study. Only 7 (4%) of 159 random sample sera reacted positively with GST. Received: 22 November 1997 / Accepted: 26 March 1998     

Peptide Microarray Analysis of In Silico-Predicted Epitopes for Serological Diagnosis of Toxoplasma gondii Infection in Humans

Toxoplasma gondii infections occur worldwide in humans and animals. In immunocompromised or prenatally infected humans, T. gondii can cause severe clinical symptoms. The identification of specific epitopes on T. gondii antigens is essential for the improvement and standardization of the serological diagnosis of toxoplasmosis. We selected 20 peptides mimicking linear epitopes on GRA1, GRA2, GRA4, and MIC3 antigenic T. gondii proteins in silico using the software ABCpred. A further 18 peptides representing previously published epitopes derived from GRA1, SAG1, NTPase1, and NTPase2 antigens were added to the panel. A peptide microarray assay was established to prove the diagnostic performance of the selected peptides with human serum samples. Seropositive human serum samples (n = 184) were collected from patients presenting with acute toxoplasmosis (n = 21), latent T. gondii infection (n = 53), and inactive ocular toxoplasmosis (n = 10) and from seropositive forest workers (n = 100). To adjust the cutoff values for each peptide, sera from seronegative forest workers (n = 75) and patients (n = 65) were used. Univariate logistic regression suggested the significant diagnostic potential of eight novel and two previously published peptides. A test based on these peptides had an overall diagnostic sensitivity of 69% (100% in ocular toxoplasmosis patients, 86% in acutely infected patients, 81% in latently infected patients, and 57% in seropositive forest workers). The analysis of seronegative sera performed with these peptides revealed a diagnostic specificity of 84%. The results of our study suggest that the use of a bioinformatic approach for epitope prediction in combination with peptide microarray testing is a powerful method for the selection of T. gondii epitopes as candidate antigens for serological diagnosis.     

Induction of tolerance toToxoplasma gondii in newborn mice by maternal antibody

To develop an animal model for analysing the suppressed immune response toToxoplasma gondii in newborn humans with congenital toxoplasmosis, newborn mice from chronically infected mothers were inoculated intraperitoneally with bradyzoites of an avirulent strain. The newborn mice with maternalToxoplasma antibodies showed a marked delay in the production ofToxoplasma antibodies when infected after birth. Many mice (11/13; 85%) developed a state of tolerance toT. gondii after disappearance of the maternal antibody, demonstrable by the absence ofToxoplasma antibody in their sera despite the fact that they were infected. The duration of tolerance differed between individuals, with two mice showing the longest tolerant state of 8 weeks. This murine model might be suitable for analysing the mechanism of suppressed immune response toT. gondii that has been observed in many human cases of congenital toxoplasmosis.     

Identification of antigens diagnostic for European isolates ofBabesia equi by two-dimensional electrophoresis and Western blotting

A lysate ofBabesia equi-infected erythrocytes (USDA strain) was separated by two-dimensional electrophoresis and anlysed by Western blotting. Nine major antigens or antigen groups with mol. wts. ranging from 43 to 19 kDa were recognized by sera from horses experimentally infected with the USDA strain. Four antigens or antigen groups were also recognized by some or all sera from horses infected withB. caballi or not infected withBabesia spp. Of the remaining five antigens, four were recognized by all sera from field-infected horses from Europe. Thus four antigens with mol. wts. of 33, 31, 19 and 20 kDa were identified as diagnostic antigens for European isolates ofB. equi. None of the antigens diagnostic for European isolates was recognized by sera from field-infected horses from Brazil.     

Dirofilaria immitis: Detection of parasite-specific antigen by monoclonal antibodies in glomerulonephritis in infected dogs

For the identification of circulating parasite antigens associated with immune-complex glomerulone-phritis in dogs infected withDirofilaria immitis, monoclonal antibodies (mAbs) were generated against adult worms. A total of 11 mAbs were selected for cloning because of their high productivity and their lack of cross-reactivity withToxocara canis in indirect immunofluorescence tests. The ability of mAbs to detect circulating antigens was examined using an antigen-capture enzyme-linked immunosorbent assay. Of the 11 mAbs, only NAK-1, an IgG2_a mAb, was capable of detecting circulating antigens in 75% of infected dogs. However, this mAb was highly species-specific in its detection of circulating antigens, since sera from dogs infected with other nematode parasites were negative. Furthermore, the mAb detected antigens at the same glomerular sites in which IgG and/or C3 were deposited. The antigen deposits were observed along capillaries and/or in mesangial cells. The epitope recognized by this mAb is probably a carbohydrate, as it remained stable for 1 h at 100° C and was sensitive to periodate treatment. Two bands of 62 and 26 kDa, respectively, were detected on Western blots by the mAb when sera from dogs infected withD. immitis were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted.     

Susceptibility of heat shock protein 70.1-deficient C57BL/6 J,wild-type C57BL/6 J and A/J mice to Trypanosoma congolense infection

Toxoplasma gondii is a protozoan parasite with a worldwide distribution. In both sheep and humans, if the parasite is encountered during pregnancy, fetal infection and abortion can occur. Therefore, Toxoplasma infection in sheep has a major economic impact upon sheep farming. Clinically, there is a need to distinguish recent (acute) infections from longstanding (chronic) infections. However, current serological techniques, such as detection of anti-T. gondii IgG, cannot discriminate between acute and chronic infections. Increasing immunoglobulin avidity is a good determining factor of how recent an infection is. In this study, we describe the application and validation of a T. gondii IgG avidity ELISA, based on the use of an affinity-purified, native T. gondii P30 antigen. The assay was used to examine sera from eight sheep experimentally infected with T. gondii and found that all seroconverted within 21 days post-infection (p.i.), beginning with avidities that were initially low but that increased over time, with all sheep reaching high IgG avidity within 10 weeks p.i. In addition, sera from clinically healthy but T. gondii-seropositive lambs and ewes and seropositive ewes with a history of abortion were also subjected to a preliminary serological investigation. High IgG avidities were found in 80% of the seropositive lambs, in 90% of the clinically healthy ewes and in 97% of the ewes with abortion problems. These findings indicate that the animals had most likely contacted the parasite a longer time ago.     

The infection-stage-related IgG response to Toxoplasma gondii studied by immunoblotting

The technique of SDS-polyacrylamide gel electrophoresis and immunoblotting was used to study the evolution of the IgG antibody response to Toxoplasma gondii antigens in sequential sera of acutely infected patients. The results show that the IgG immunoblot pattern can be a useful marker for the stage of T. gondii infection. A 35-kD antigen elicited the first IgG response soon after exposure. During the course of infection additional bands appeared consecutively, following a constant sequence, to evolve to a late-stage-specific pattern with about 13–15 major bands. This pattern, which was reached at least 4 months after infection, was also found in the immunoblots of 28 patients with a chronic (latent) T. gondii infection.     

Unspecific Reactivity of IgM Directed Against the Low-Molecular-Weight Antigen of<Emphasis Type="Italic"> Toxoplasma gondii</Emphasis>

During routine serological survey, eight patients (5 pregnant women, 3 grafted patients) were positive for Toxoplasma gondii-specific IgM by enzyme-linked immunoassay but negative by a simultaneously performed immunosorbent agglutination assay. No clinical or biological symptoms of toxoplasmosis were observed later, despite the absence of treatment. Only one IgM-reactive band, which corresponded to the low-molecular-weight antigen of Toxoplasma gondii, was observed by Western blotting of these patients' sera. Dot blotting of lipid extracts of Toxoplasma gondii demonstrated that this reactivity was directed against sphingolipids or ceramides. This IgM positivity, which is unrelated to acute toxoplasmosis, raises strong concerns about the possibility of misleading results of this test in the diagnosis of toxoplasmosis in humans.     

Serological Evidence of Human Infection with the Protozoan Neospora caninum

Neospora caninum is a protozoan parasite that is closely related to Toxoplasma gondii. Dogs are a definitive host. Prior to its discovery in 1988, N. caninum infection in animals was often mistakenly diagnosed as toxoplasmosis. Neosporosis in animals is characterized by encephalitis, abortion, and other conditions that clinically and pathologically resemble toxoplasmosis. The potential of N. caninum to infect humans is unknown. Therefore, evidence of human exposure to this parasite was sought by screening for antibodies in blood donors by indirect fluorescent antibody (IFA) tests and immunoblotting. Of 1,029 samples screened, 69 (6.7%) had titers of 1:100 by IFA testing. Fifty of the 69 (72%) sera that were positive for N. caninum were also negative for a closely related protozoan pathogen of humans, T. gondii. Immunoblot analysis confirmed the specificity of the positive sera for N. caninum antigens, with several sera recognizing multiple Neospora antigens with molecular masses similar to those of antigens recognized by monkey anti-N. caninum serum. An immunodominant antigen of approximately 35 kDa was observed with 12 sera. These data provide evidence of human exposure to N. caninum, although the antibody titers in healthy donors were low. The significance of human exposure to, and possible infection with, this parasite is unknown and warrants further study.     

Animals are key to human toxoplasmosis

Toxoplasma gondii is an extremely sucessfull protozoal parasite which infects almost all mamalian species including humans. Approximately 30% of the human population worldwide is chronically infected with T. gondii. In general, human infection is asymptomatic but the parasite may induce severe disease in fetuses and immunocompromised patients. In addition, T. gondii may cause sight-threatening posterior uveitis in immunocompetent patients. Apart from few exceptions, humans acquire T. gondii from animals. Both, the oral uptake of T. gondii oocysts released by specific hosts, i.e. felidae, and of cysts persisting in muscle cells of animals result in human toxoplasmosis. In the present review, we discuss recent new data on the cell biology of T. gondii and parasite diversity in animals. In addition, we focus on the impact of these various parasite strains and their different virulence on the clinical outcome of human congenital toxoplasmosis and T. gondii uveitis.     

Recombinant proteins in the diagnosis of toxoplasmosis

Kotresha D, Rahmah N. Recombinant proteins in the diagnosis of toxoplasmosis. APMIS 2010; 118: 529–42. Toxoplasma gondii is an important human pathogen with a worldwide distribution. It is primarily of medical importance for pregnant women and immunocompromised patients. Primary infection of the former is often associated with fetal infection, which can lead to abortion or severe neonatal malformation. Immunocompromised patients are at risk of contracting the severe form of the disease that may be fatal. Thus, detection of T. gondii infection with high sensitivity and specificity is crucial in the management of the disease. Toxoplasmosis is generally diagnosed by demonstrating specific immunoglobulin M (IgM) and IgG antibodies to toxoplasma antigens in the patient’s serum sample. Most of the commercially available tests use T. gondii native antigens and display wide variations in test accuracy. Recombinant antigens have great potential as diagnostic reagents for use in assays to detect toxoplasmosis. Thus in this review, we address recent advances in the use of Toxoplasma recombinant proteins for serodiagnosis of toxoplasmosis.     

Estimation of the avidity of immunoglobulin G for routine diagnosis of chronicToxoplasma gondii infection in pregnant women

Present serological methods differentiate poorly between acute and chronic toxoplasmosis in pregnant women, particularly when immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies toToxoplasma gondii are present simultaneously. In the present study, a simple test for discriminating between high-avidity antibodies, which are usually present in chronic infections, and low-avidity antibodies, typical of acute infection, was evaluated. Sera were evaluated forToxoplasma gondii antibodies using a commercial enzyme immunoassay, but a duplicate well was washed in 6M urea to disrupt lowavidity complexes. Results are expressed as the percentage of antibodies resisting elution by urea. Equivocal sera (n=493) containing both IgG and IgMToxoplasma gondii antibodies from 309 pregnant women whose status as chronically or acutely infected had been independently determined using standard methods were evaluated for antibody avidity. A value of >35% elution-resistant antibodies was always associated with chronic infection and could absolutely exclude a recent (<3 months) infectious incident. Values of <35% require repeat testing four weeks later to confirm the patient's status, since a proportion of individuals with chronic toxoplasmosis maintain low-avidity antibodies over long periods. This inexpensive, simple method can provide reassurance to clearly chronically infected individuals and avoids the need for repeated testing in these cases.     

Transplacental transmission in cattle: is Toxoplasma gondii less potent than Neospora caninum?

We compared the transplacental-transmission ability of Toxoplasma gondii and Neospora caninum in cattle. One uninfected pregnant heifer served as control, while three were inoculated with N. caninum K9WA strain and four with T. gondii RH strain at their midgestational period. Both infected groups showed clinical signs and antibodies either to N. caninum or T. gondii, while the control animal was normal. Two (50%) Toxoplasma dams aborted on days 6 and 11 postinoculation. T. gondii tachyzoites were found in various organs of those dams that had abortions but not in their fetuses. Two Neospora dams did not abort but gave birth to subclinically infected calves. The remaining two Toxoplasma dams and one from Neospora group became recumbent. Those two dams and their fetuses showed disseminated Toxoplasma DNA, but no Neospora DNA was found. Our findings suggest that maternal toxoplasmosis could be a cause of abortion and congenital toxoplasmosis in cattle, especially when they are infected by virulent strains.     

Antigenic characterisation of monoclonal antibodies againstSarcocystis muris by Western blotting and immuno-electron microscopy

The antigenic specificities of 13 monoclonal antibodies (mAbs) raised againstSarcocystis muris cystozoites were examined by Western blotting and immuno-electron microscopy against homologousS. muris and heterologousS. gigantea, S. tenella, S. arieticanis, S. capracanis, S. miescheriana andToxoplasma gondii antigens. Four mAbs reacted in Western blots againstS. muris antigens: SM-4 and-17 recognized single antigenic bands (31000 and 34000 MW, respectively) and SM-2 and-3 reacted against multiple bands (ranging from 12500–30000 and 13000–50000 MW, respectively). Similar antigens were also recognized by polyclonal immune sera from chronically infected mice. None of the mAbs cross-reacted with heterologousSarcocystis spp. orT. gondii. Ultrastructural studies performed with colloidal-gold conjugates demonstrated that three mAbs reacted with specific antigenic elements inS. muris cystozoites: SM-3 and-4 labelled pellicular determinants and SM-19 labelled micronemes. None of the mAbs crossreacted with heterologousSarcocystis spp., whereas polyclonal immune sera from chronically infected sheep, goats and pigs cross-reacted with a variety of antigens in allSarcocystis spp. except the primary cyst-wall determinants.Abbreviations cz Cystozoite - fi tibril - gs ground substance - mi micronemes - pe pellicle - pw primary cyst wall - rh rhoptries     

Genotyping of samples from German patients with ocular,cerebral and systemic toxoplasmosis reveals a predominance of Toxoplasma gondii type II

Toxoplasmosis is an important zoonosis transmitted from animals to humans world-wide. In order to determine Toxoplasma gondii genotypes in individuals living in Germany and to compare findings with those in animals, we analysed nine independent and unlinked genetic markers (nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) by PCR-RFLP in 83 archived T. gondii-positive DNA samples from patients with ocular toxoplasmosis (n = 35), toxoplasmic encephalitis (n = 32), systemic toxoplasmosis after bone-marrow transplantation (n = 15) and congenital toxoplasmosis (n = 1). In 46 of these 83 samples the presence of T. gondii DNA was confirmed by conventional end-point PCR. Among these, 17 T. gondii-positive samples were typed at all nine loci. The majority (15/17, 88.2%) of these samples were of T. gondii type II (i.e., including both, the Apico type II and Apico type I variants). In addition, in one sample a T. gondii type II/type III allele combination and in another sample a T. gondii genotype displaying type III alleles at all markers was observed. In the remaining 11 samples, in which T. gondii could only be partially typed, exclusively type II (n = 10) or type III (n = 1) alleles were observed. Results of the present study suggest that the majority of patients in Germany are infected with type II T. gondii regardless of the clinical manifestation of toxoplasmosis. This finding is in accord with the predominance of type II T. gondii in oocysts isolated from cats and in tissues of other intermediate hosts in Germany.     

Application of the specific protein-fixation test in toxoplasmosis

Summary By means of fixation of the antiserum protein with the paper-sorbed antigen (PF) a study was made of the sera of rabbits and rats infected withToxoplasma gondii as well as of the sera obtained from persons with a preliminary diagnosis of toxoplasmosis. The results were positive in about 90% of the experiments on the infected animals and in 50% of the persons with the clinically suspected toxoplasmosis.PFT is recommended for immunological studies of toxoplasmosis.(Presented by Active Member AMN SSSR, N. N. Zhukov-Verezhnikov) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 59, No. 1, pp. 104–107, January, 1965     

Antibody responses of experimentally infected lambs to antigens collected during in vitro maintenance of the adult,metacestode or oncosphere stages ofTaenia hydatigena andTaenia ovis with further observations on anti-oncospheral antibodies

The antigenicity and specificity of crude antigens collected during the in vitro maintenance ofTaenia hydatigena andT. ovis, excretory/secretory (ES) antigens, were assessed in a peroxidase microenzyme-linked immunosorbent assay (ELISA), using sera from lambs given experimental monospecific infections withT. hydatigena, T. ovis, Echinococcus granulosus orFasciola hepatica.ES antigens of larval cysts ofT. ovis andT. hydatigena were less reactive than those of adult or oncosphere stages. Strong interspecific cross-reactions occurred between all antigen preparations, and these antigens offered no better specificity than crude somatic extracts. IgG₁ was the major immunoglobulin detected in sera from lambs experimentally infected withT. ovis orT. hydatigena using antigens prepared from sonicated oncospheres. Discrete peaks of anti-oncospheral antibodies were detected following initial and challenge infections with eggs (whether the homologous or heterologous species), when sera were assayed with a PBS sonicate or an ES antigen from oncospheres. However, when oncospheres solubilised with sodium deoxycholate were used, the antibody response was prolonged and resembled that reported previously when somatic extracts of adult and metacestode stages were used as antigen. The results showed that oncospheres share antigens in common with other life-cycle stages, but also support the notion that they may possess some unique stage-specific antigenic determinants.     

Seroprevalence and risk factors of Toxoplasma gondii infection in invasive raccoons (Procyon lotor) in Central Europe

Toxoplasma gondii is an obligate intracellular protozoan that causes toxoplasmosis in warm-blooded animals. Most mammals, including humans, can become intermediate host, resulting in subclinical infection or even death. Generally, there is limited information on the epidemiology of T. gondii of game species in Germany. As omnivores, raccoons, which are particularly widespread and abundant in Germany, are particularly exposed to infection the parasite. Here, we report the seroprevalence of T. gondii antibodies from 15 study sites located in Luxembourg and Germany. Using the indirect modified agglutination test (MAT), 170 (37.4%; 95% CI: 33.0–41.9) out of 454 raccoons were surveyed to be T. gondii seropositive. While values ranged from 19.0% to 53.3%, there was no significant difference in seroprevalence between study areas. Animal weight had a strong influence on the presence of T. gondii antibodies in raccoon sera, with heavier animals more likely to be seropositive. Our results show that T. gondii infection is widespread in central European raccoons, suggesting a high degree of ecosystem circulation of the parasite.

     

Recognition of Toxoplasma gondii excreted and secreted antigens by human sera from acquired and congenital toxoplasmosis: identification of markers of acute and chronic infection.

While the serological response to somatic antigens of Toxoplasma gondii is currently analysed, little information is available on the antibody response to the antigens excreted and secreted by tachyzoïtes (ESA). This serological study is focused on the immune response towards these antigens which were released by the parasites in cell-free culture medium. Human sera corresponding to ''acute'', ''subacute'' and ''chronic'' acquired infection and sera from infected newborns and from their mothers were analysed by radio-immunoprecipitation with 35S methionine-labelled ESA and with radio-iodinated membrane antigens followed by polyacrylamide gel electrophoresis. In chronic toxoplasmosis, IgG antibodies recognized among ESA major 108, 97, 86, 60, 57, 42, 39 and 28.5 kD antigens; the 108-97 kD doublets and the 28.5 kD antigen seemed characteristic of the chronic phase of toxoplasmosis. In acute infection, IgM antibodies to the 97 kD antigen, the first to appear, seem to contitute good markers of early acute infection. The comparative study of antibody response to membrane antigens showed that, in chronic toxoplasmosis, human sera recognized four antigens of 43, 35, 30 and 22 kD and that, in acute toxoplasmosis, they first recognized the 43 and 30 kD antigens. The serological evolution in congenital toxoplasmosis was the same as in acquired infection. In some cases, the serological profile of the newborn was different from that of his mother, with an additional antibody response to a 170 kD antigen. This study demonstrates in human toxoplasmosis an early, intense and characteristic antibody response against ESA, suggesting that the use of these antigens could lead in the future to improved diagnostic tests.     

Toxoplasma gondii surface antigen-1 in sera of HIV-infected patients as an indicator of reactivated toxoplasmosis

The major surface antigen from the proliferative form ofToxoplasma gondii (P-30 or SAG-1) was chosen as a target for exploration ofToxoplasma gondii reactivation in sera from immunocompromised patients. Samples were obtained from 37 HIV-infected subjects with lymphocyte levels of CD4+ <200/mm³. The prevalence of IgG antibodies toToxoplasma gondii was 64.9 %. Ten patients had clinical symptoms of reactivated toxoplasmosis; eight of these hadToxoplasma encephalitis. The SAG-1 epitopes were found as circulating antigen in five cases with an immunocapture enzyme immunoassay (EIA). The EIA was improved with an IgG1 monoclonal antibody to SAG-1 and a streptavidinbiotin amplification. The sensitivity, specificity and positive predictive value were 30, 92 and 60 %, respectively. The SAG-1 levels were compared with different biological parameters such as HIV p24 antigen, ₂ microglobulin, CD4+ cell count and IgG antibodies toToxoplasma gondii. The levels of SAG-1 in these patients were significantly higher than those in the 75 healthy control persons with or without a chronicToxoplasma gondii infection. Therefore, SAG-1 may be involved as a marker of reactivated toxoplasmosis in HIV-infected patients.