IgG Western Blot for Confirmatory Diagnosis of Equivocal Cases of Toxoplasmosis by EIA-IgG and Fluorescent Antibody Test

The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.     

The use of commercial immunoenzyme kits for the diagnosis of toxoplasmosis

The results of comparative study of sera obtained from donors and from several groups of patients with suspected toxoplasmosis are presented. The study has been carried out with the use of commercial enzyme immunoassay (EIA) kits: Toxoplasma gondii IgG EIA and Toxoplasma gondii IgM EIA manufactured by Labsystems (Finland), Sevatest ELISA IgG Toxo Micro I manufactured by Sevac (Czechoslovakia). Statistical processing of the results has confirmed the identity of these kits. The necessity of using evaluation criteria (the separation point, the scale for the interpretation of results) when working with the Sevac kits is emphasized. Comparative evaluation of antibody profiles in the sera under test suggests that the titer less than 1:1600 should be regarded as the separation point for these kits. IgM antibodies to T. gondii have been found only in 22% of patients with high titers of IgG antibodies.     

Toxoplasma gondii: DNA vaccination with genes encoding antigens MIC2, M2AP, AMA1 and BAG1 and evaluation of their immunogenic potential

A combination of antigenic regions of microneme proteins have been previously reported as being protective against chronic toxoplasmosis. In this work, we evaluated immune responses induced by immunizing BALB/c and C57BL/6 mice intradermally with plasmid DNA encoding the protein sequences of Toxoplasma gondii AMA1, MIC2, M2AP and BAG1. Mice immunized with the AMA1 gene developed high levels of serum IgG2a and c antibodies as well as cellular immune responses associated with IFN-gamma synthesis suggesting a modulated Th1 type of response. Immunization with the AMA1 gene resulted in a partial but significant protection against the acute phase of toxoplasmosis compared to MIC2, M2AP and BAG1 genes. Therefore, the AMA1 gene appears to generate a strong specific immune response and also provides effective protection against toxoplasmosis more than the MIC2, M2AP and BAG1 genes.     

Immune responses of different mouse strains after challenge with equivalent lethal doses of Toxoplasma gondii

Most immunological studies that utilize different strains of inbred mice following T. gondii infection fail to compensate for differences in host susceptibility to the size of the parasite innoculum. To address this concern, susceptible C57BL/6 and resistant CBA/J mice were orally infected with either an equivalent 50% lethal dose (LD50) of brain cysts of the 76K strain of T. gondii (15 cysts in C57BL/6, 400 cysts in CBA/J) or the same dose of parasites in each mouse strain. C57BL/6 mice receiving 400 cysts (LD50 of CBA/J mice) died post infection, whereas CBA/J mice that received 15 cysts (LD50 of C57BL/6 mice) survived. Parasite loads in the brains and serum Toxoplasma-specific IgG1 titers of LD50-infected C57BL/6 mice were significantly higher than those in LD50- or 15 cysts-infected CBA/J mice, whereas splenocyte proliferation to Toxoplasma antigen and the percentage of CD8 alpha+ T cells were reduced in LD50-infected C57BL/6 mice. In contrast, serum IgG2a and IgM titers, the percentage of gamma delta T cells and IFN-gamma expression of spleen of LD50-infected CBA/J mice were higher than those of either 15 cysts-infected CBA/J mice or LD50-infected C57BL/6 mice. These observations demonstrate that the immune response between LD50-infected C57BL/6 and CBA/J mice was more prominent when compared to C57BL/6 or CBA/J mice receiving the same parasite inoculum. These observations would suggest that caution must be excersized in the planning and interpretation of data when the size of the parasite inoculum has not been adjusted for mouse strain.     

Maintained seroprevalence of toxoplasmosis among the residents of Jeju island, Korea

Seroepidemiological status of toxoplasmosis among the residents of Jeju island was surveyed and evaluated by ELISA with crude extract of Toxoplasma gondii. The sera of 2,348 residents (male 1,157 and female 1,191) were collected and checked for the IgG antibody titers, which showed 13.2% positive rate (309 sera). The positive rates were increasing gradually according to the age from 4.3% in teenage to 20.6% in seventies. The positive rates were significantly different between the sex by 16.2% for male and 10.2% for female (P<0.05). This positive rate of toxoplasmosis in Jeju island residents is regarded relatively higher than any other regions of Korea. And the high positive rate may be maintained continuously among Jeju island residents without any clear reasons until now but due to some parts peculiar socio-cultural tradition of Jeju island. Therefore, it is necessary to study further the epidemiology of toxoplasmosis of Jeju island.     

Changes in blastogenic responses and antibody titers of mice infected with Toxoplasma gondii]

This study was performed to observe the cell-mediated and humoral immune responses in mice which were infected with Beverley, Fukaya and ME49 strain of Toxoplasma gondii, respectively. The blastogenic responses of splenocytes using [3H]-thymidine and serum antibody titers were measured weekly up to 10 weeks after infection. The blastogenic responses of splenocytes treated with concanavalin A and Toxoplasma lysate were significantly declined in the 3 strain groups as compared with the non-infected group (p less than 0.05), however lipopolysaccharide-treated blastogenic responses were not significantly different between infected and non-infected groups. The serum IgG antibody titers in the three infected groups increased from 2 weeks after infection, and the serum IgM antibody titers increased until 4 weeks after infection. No significant differences were revealed in blastogenic responses and serum antibody titers among the 3 groups. The present study suggested that cell-mediated immune responses were involved in T. gondii infected mice and blastogenic responses of T lymphocytes were inhibited in acute T. gondii infection.     

Seroprevalence of Toxoplasmosis in the Residents of Cheorwon-gun, Gangwon-do, Korea

The seroepidemiological status of toxoplasmosis was surveyed among the residents of Cheorwon-gun, Gangwon-do by means of ELISA using a crude extract antigen of Toxoplasma gondii. The sera of 1,661 adult residents (866 males and 795 females) were collected and checked for IgG antibody titers, which showed 17.0% positive rate (282 sera). The positive rate was significantly different between the sex; 20.6% for males and 13.1% for females (P<0.05). The positive rates were higher in fifties of males (28.7%) and forties of females (20.0%). This positive rate of toxoplasmosis in Cheorwon-gun residents is regarded as the highest among the surveys of different geographical regions of Korea. This high positive rate may due in part to peculiar geographical locality of the surveyed area near the naturally well preserved demilitarized zone (DMZ) or presumably consumption of the pork imported from high endemic nations. Therefore, it is necessary to study further the epidemiology of toxoplasmosis in Cheorwon-gun.     

Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps

A key feature of helminth infections is the induction of strong Th2-biased immune responses in their hosts. We have previously found that Th2-like responses mediate susceptibility to the helminth parasite Taenia crassiceps, probably by inhibiting Th1 responses required for the development of protective immunity against this parasite. Here we show that mice lacking interleukin-12p35 (IL-12p35-/-) following T. crassiceps infection, failed to mount a Th1 response, but developed a strong Th2-type response, produced higher levels of IgG1, IgE, interleukin-4, interleukin-5 as well as interleukin-13 than wild-type mice, and became highly susceptible to the larval stage of this cestode. In contrast, similarly-infected CD40 deficient BALB/c mice (CD40-/-) displayed impairment of both Th1 and Th2-type responses associated with low levels of interferon-gamma as well as IgE, interleukin-4, interleukin-5 and interleukin-13, but efficiently controlled T. crassiceps infection. Together, these findings suggest a detrimental role for Th2-biased responses during the larval stage of T. crassiceps infection. Furthermore, they also suggest a pivotal role for CD40 in developing Th2-type responses.     

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.     

Immune response to Toxoplasma gondii--analysis of suppressor T cells in a patient with symptomatic acute toxoplasmosis

Unresponsiveness of antigen-dependent (Toxoplasma-specific and purified protein derivative of tuberculin [PPD]-specific) T-cell proliferative responses of peripheral blood leukocytes (PBL) was observed in a patient with symptomatic acute toxoplasmosis. The immunosuppression of T-cell responses was mediated by Leu 1+, Leu 2a+, and Leu 3a- suppressor T cells that were induced by Toxoplasma gondii antigen and suppressed both Toxoplasma-specific and PPD-specific PBL T-cell responses from a patient with chronic toxoplasmosis when PBL of these patients were mixed and cocultured in vitro. Participation of class II molecules of HLA in Toxoplasma-specific proliferative T-cell responses and activation of suppressor T cells was examined by using monoclonal antibodies specific for HLA-DR and HLA-DQ molecules. Anti-HLA-DQ monoclonal antibody released the suppressive activity, while anti-HLA-DR monoclonal antibody inhibited Toxoplasma-specific T-cell responses. Thus, the suppressive effect of PBL from a patient with acute toxoplasmosis on antigen-dependent PBL T-cell responses from a patient with chronic toxoplasmosis was mediated by HLA-DQ molecules. By contrast, Toxoplasma-specific T-cell responses were activated by HLA-DR molecules (presumably present on antigen-presenting cells).     

An opposite role is exerted by the acarian Myocoptes musculinus in the outcome of Toxoplasma gondii infection according to the route of the protozoa inoculation

Infection with Toxoplasma gondii leads to a Th1 immune response. Alternatively, the acarian Myocoptes musculinus induces a disease in BALB/c mice that involves Th2 immune mechanisms. In this study, we investigated whether infestation by M. musculinus induces Th2 immune response in C57BL/6 mice and if this response influences the T. gondii-induced Th1 response when mice are inoculated by intraperitoneal or oral route. The animals were infected with M. musculinus and one month later with T. gondii ME-49 strain and the survival and immune response were monitored. The co-infected animals displayed higher mortality rate and the spleen cells showed a decreased IFN-gamma and elevated IL-4 and IL-5 production. These changes were associated with severe pneumonia and wasting condition. On the other hand, when mice were orally infected with 100 T. gondii cysts, co-infection prolonged the survival rates and ameliorated intestinal lesions in association with a significant drop in IFN-gamma levels in sera. These results indicate the interference of Th2 response induced by M. musculinus in a T. gondii-induced Th1 response. Altogether, these data demonstrate the profound interactions between the immune response induced against unrelated organisms T. gondii and M. musculinus, and suggest that this type of interactions may impact clinical disease.     

Toxoplasma gondii: the vaccine potential of three trivalent antigen-cocktails composed of recombinant ROP2, ROP4, GRA4 and SAG1 proteins against chronic toxoplasmosis in BALB/c mice

Toxoplasmosis is one of the world's most widespread zoonoses caused by protozoan parasite Toxoplasma gondii. The development of an effective vaccine for controlling toxoplasmosis is an extremely important issue due to the serious clinical and veterinary outcomes of this parasitosis. The objective of this study was evaluation of vaccine potential of three trivalent subunit recombinant vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 against chronic toxoplasmosis in BALB/c (H-2(d)) mice. All tested vaccines provided a partial protection against challenge with tissue cysts of the low virulence DX T. gondii strain, but the strongest level of protection was induced by the mixtures of both rhoptry proteins (rROP2 and rROP4) administered with the dense granule rGRA4 antigen or the main surface rSAG1 protein. The average parasite burden in these groups of vaccinated BALB/c mice was reduced by 84% and 77%, respectively, compared to the control PBS-injected animals. The vaccine-induced protection was correlated with the development of cellular and humoral immune responses demonstrated by the antigen-specific in vitro proliferation of spleen cells, the specific antigen-induced in vitro synthesis of Th1-type cytokines, IFN-γ and IL-2, and the generation of the high titers of systemic antigen-specific IgG1 and IgG2a antibodies. This study completed and confirmed our earlier investigations in C3H/HeJ (H-2(k)) and C57BL/6 (H-2(b)) mouse strains on the utility of the tested trivalent recombinant antigen-cocktails as potential vaccines against chronic toxoplasmosis and showed that particularly rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 protein-combinations are very effective in the development of a high level of protection irrespective of the genetic backgrounds and innate resistance to toxoplasmosis of the laboratory mice. It makes these two mixtures of recombinant antigens very promising for further experiments.     

Immune Responses of NIH Mice Infected with Avirulent and Virulent Strains of Plasmodium chabaudi adami Single and Mixed Infections

An understanding of the nature of the immune response to asexual erythrocytic stages of malaria parasites will facilitate vaccine development by identifying which responses the vaccine should preferentially induce. The present study examined and compared the immune responses of NIH mice in either single or mixed infections with avirulent (DK) or virulent (DS) strains of Plasmodium chabaudi adami using the ELISA test for detecting and measurement of cytokines and antibody production. In both single and mixed infections, the study showed that both cell- and antibody-mediated responses were activated. In all experiments, an early relatively high level of IFN-γ and IgG2a during the acute phase of the infection, and later elevation of IL-4 and IgG1, suggested that there was a sequential Th1/Th2 response. However, in the avirulent DK strain infection a stronger Th1 response was observed compared to the virulent DS strain-infection or in mixed infections. In the virulent DS infection, there was a stronger Th2 response compared to that in the DK and mixed infections. The faster proliferation rate of the virulent DS strain compared to the DK strain was also evident.     

Seroprevalence of toxoplasmosis in the residents of Cheju island, Korea

This study was performed to evaluate the epidemiological status of toxoplasmosis among the residents of Cheju island. The sera of local students from 18 high schools (boys 2110, girls 2460) and those of adults (474 admitted to Cheju Chungang General Hospital) were collected and checked for the IgG antibody titers against Toxoplasma gondii. Serum samples collected from both the students and adults showed sero-positive rate of 5.5% and 12.9%, respectively. Although the rates were not significantly different between the sexes (5.4% for the boys and 5.5% for the girls attending school), the geographical difference showed a significant difference between the urban (4.6-6.9%) and rural areas (5.6-8.8%) (p < 0.05). Based on the high positive rates, it should be necessary to control toxoplasmosis in Cheju island.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up regulation of Th1-dominant polarization in BALB/c mice

The effects of graded doses of a chemically standardized aqueous alcoholic (1:1) root extract (AGB) of Withania somnifera on the immune system of SRBC immunized BALB/c mice were investigated. Mice were administrated AGB orally for 15 days. AGB stimulated cell mediated immunity, IgM and IgG titers reaching peak value with 30 mg/kg b.wt. Flow cytometric analysis of lymphocyte surface markers of T cells (CD3(+), CD4(+) and CD8(+)) and B cells (CD19(+)) indicated prominent enhancement in proliferation and differentiation of lymphocytes. The extract selectively, induced type 1 immunity because it guided enhanced expression of T helper cells (Th)1 cytokines interferon (IFN)-gamma and interleukin (IL)-2 while Th2 cytokine IL-4 observed a moderate decline. Confirmation of Th1 polarization was obtained from augmented levels of IgG2a over IgG1 in the blood sera of AGB treated groups. Withanolide-A, a major constituent of AGB appeared responsible for Th1 skewing effect of the extract as it significantly increased the levels of Th1 cytokines, decreased moderately IL-4 and significantly restored the selective dexamethasone inhibition of Th1 cytokines in mouse splenocytes cultures in vitro. In addition, AGB also strongly activated macrophage functions ex vivo and in vitro indicated by enhanced secretion of nitrite, IL-12 and TNF-alpha. In contrast IL-10 remained unchanged again suggesting that AGB critically influenced Th1 profile of the cytokines. The studies suggested that AGB supports predominantly Th1 immunity with increase in macrophage functions. The standardized root extract of no toxicological consequences might therefore, find useful applications against the intracellular pathogens and in the management of immune suppressed diseases.     

Intranasal Immunisation with Recombinant Toxoplasma gondii Actin Partly Protects Mice against Toxoplasmosis

Toxoplasma gondii is a ubiquitous protozoan intracellular parasite, the causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. Actin is a highly conserved microfilament protein that plays an important role in the invasion of host cells by T. gondii. This study investigated the immune responses elicited by BALB/c mice after nasal immunisation with a recombinant T. gondii actin (rTgACT) and the subsequent protection against chronic and lethal T. gondii infections. We evaluated the systemic response by proliferation, cytokine and antibody measurements, and we assessed the mucosal response by examining the levels of TgACT-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes. Parasite load was assessed in the liver and brain, and the survival of mice challenged with a virulent strain was determined. The results showed that the mice immunised with rTgACT developed high levels of specific anti-rTgACT IgG titres and a mixed IgG1/IgG2a response with a predominance of IgG2a. The systemic immune response was associated with increased production of Th1 (IFN-γ and IL-2), Th2 (IL-4) and Treg (IL-10) cytokines, indicating that not only Th1-type response was induced, but also Th2- and Treg-types responses were induced, and the splenocyte stimulation index (SI) was increased in the mice immunised with rTgACT. Nasal immunisation with rTgACT led to strong mucosal immune responses, as seen by the increased secretion of SIgA in nasal, vaginal and intestinal washes. The vaccinated mice displayed significant protection against lethal infection with the virulent RH strain (survival increased by 50%), while the mice chronically infected with RH exhibited lower liver and brain parasite loads (60.05% and 49.75%, respectively) than the controls. Our data demonstrate, for the first time, that actin triggers a strong systemic and mucosal response against T. gondii. Therefore, actin may be a promising vaccine candidate against toxoplasmosis.     

A New IgG Immunoblot Kit for Diagnosis of Toxoplasmosis in Pregnant Women

The determination of the accurate immune status of pregnant women is crucial in order to prevent congenital toxoplasmosis. Equivocal results with conventional serological techniques are not uncommon when IgG titers are close to the cut-off value of the test, so that a confirmatory technique is needed. For this purpose, we developed a homemade immunoblot (IB) using soluble extract of Toxoplasma gondii tachyzoites and assessed it by testing 154 positive, 100 negative, and 123 equivocal sera obtained from pregnant women. In order to select the more valuable bands in terms of sensitivity and specificity, we used the Youden Index (YI). The highest YIs were those given by the 32, 36, 98, 21, and 33 bands. The simultaneous presence on the same blot of at least 3 bands showed a much higher YI (0.964) and was adapted as the positivity criterion. The analysis of results showed that our homemade IB correlated well with the commercial LDBIO Toxo II IgG® kit recently recommended as a confirmatory test (96.7% of concordance).     

Depletion of CD4+ T cells but not inhibition of the protective activity of IFN-gamma prevents cure of toxoplasmosis mediated by drug therapy in mice.

Toxoplasmosis is a frequent opportunistic infection in patients with AIDS. In these patients the major immune deficiencies are a severe depletion of CD4+ T lymphocytes and an impaired capacity to produce IFN-gamma. A mouse model was developed and used to study the effects that depletion of CD4+ T cells and/or inhibition of the protective activity of IFN-gamma have on the effectiveness of the drug therapy for toxoplasmosis. Infection of mice with a lethal inoculum of Toxoplasma gondii cysts followed by treatment with the hydroxynaphthoquinone 566C80 or with sulfadiazine resulted in 100% survival whereas untreated controls had 100% mortality within 15 days of infection. Administration of antiserum to IFN-gamma resulted in early death of untreated mice and in 30% mortality in those treated. Administration of mAb to CD4+ T cells followed by infection with T. gondii prevented the development of both antibody and cell-mediated immune responses against the parasite. These mice resisted the acute infection while undergoing specific treatment. Discontinuation of the treatment, however, resulted in reactivation of the infection and the majority of the animals died within 17 days of suspension of the treatment. Administration of antiserum to IFN-gamma or to CD4+ T cells 24 h but not 15 days after conclusion of the treatment also resulted in mortality. These results indicate that successful treatment of toxoplasmosis depends on the status of the immune system, particularly of CD4+ T cells. Although it is speculative to compare results obtained in mice to the situation in humans, our work suggests that restoration of a competent immune response is of crucial importance for a successful treatment of toxoplasmosis in immunocompromised individuals.