Serological survey of antibodies to Toxoplasma gondii

Toxoplasmosis is one of the most prevalent parasitic infections of man and livestock, and its transmission has usually been attributed to ingestion of undercooked or raw meat from infected livestock, with the infection rate in those animals being an important risk predictor of human disease, high in Iran and Ardabil State. During a study on this public health problem, we tested serum samples from cattle, goats, sheep and chicken from the State of Ardabil, Iran, for IgG antibodies to Toxoplasma gondii by enzyme-linked immunosorbent assay (ELISA). Antibodies to Toxoplasma gondii were found in 30 % (60 / 200) of sheep, 15 % (30 / 200) of goats and 9 %(18 / 200) of cattle, and none were found in chicken sera. Despite the differences in feeding habits of each species, the rate of infection of the animals tested could be attributed to livestock management methods, whose improvement could reduce infection.     

Seroprevalence and systemic immune biomarkers associated with Toxoplasma gondii infection in blood donors from Southern Brazil

The high seroprevalence of Toxoplasma gondii infection in Blood Banks could be a potential risk for contamination of blood recipients. The discovery of new biomarkers may help to distinguish between seropositive and seronegative donors. This study determined the seroprevalence and profile of systemic immune biomarkers associated with Toxoplasma gondii infection among blood donors from Southern Brazil. Peripheral blood was collected from 510 blood donors (52.2 % male; mean age: 36.61), 310, and 200 from Erechim, and Chapecó municipalities, respectively. Specific Toxoplasma gondii IgG and IgM antibodies were detected by Eletrochemioluminescence. Nested PCR and qPCR were performed to detect Toxoplasma gondii DNA. Twenty-seven inflammatory factors were analyzed using a high-performance Luminex assay. Among 310 blood donors from Erechim, 44.5 % (138/310) were IgM(?)/IgG(+), and 1.3 % (4/310) were IgM(+)/IgG(+), while out of 200 blood donors from Chapeco, 42.5 % (85/200) were IgM(?)/IgG(+), and 2 % (4/200) were IgM(+)/ IgG(+). We did not find Toxoplasma gondii DNA in the samples analyzed by Nested PCR and qPCR. Additionally, IgM(?)/IgG(+) donors presented higher levels of distinct systemic mediators, and were indicated to be high producers of several systemic mediators (CCL11, CCL2, CCL3, CCL4, CXCL10, IL-1β, IL-17, IFN-γ, IL-4, IL-9, IL-13, IL-10, IL-1Ra, vascular endothelial growth factor/VEGF, platelet-derived growth factor/PDGF, granulocyte–macrophage colony-stimulating factor/GM-CSF, and IL-7). However, IgM(+)/IgG(+) donors were found as high producers of CXCL8, CXCL10, CCL4, IL-1β, IL-1Ra, IL-9, IL-13, and PDGF, while IgM(?)/IgG(?) donors showed unaltered levels for the most soluble mediators evaluated. These distinct biomarker signatures might help identify potential factors to distinguish between IgM(?) and IgM(+) donors.     

Monoclonal antibodies identify new Toxoplasma gondii soluble antigens

In order to characterize Toxoplasma gondii antigens, we have produced a panel of monoclonal antibodies specific for the parasite. A total of 22 hybridomas were derived from the spleen cells of mice immunized either with a 100,000 g supernatant of a sonicate from the RH strain (called F3), or chronically infected with the Wiktor or the 76K strain. Except for one hybridoma producing an IgM, all the hybridomas derived from mice immunized with F3 produced IgG1 antibodies while those obtained from chronically infected mice produced antibodies belonging to the IgG2b, IgG2a and IgM subclasses. Western-blot analysis showed that the panel of monoclonal antibodies defines at least 7 distinct antigens or antigen families. An antigen of apparent Mw 25 kD present exclusively in the 100,000 g supernatant of the T. gondii sonicate was recognized by the majority of monoclonal antibodies derived from mice immunized with the F3 fraction. Two other antigens of apparent Mw 27 kD and 29 kD present in the soluble and insoluble fractions of the sonicate were also identified. Monoclonal antibodies against the previously described 21 kD and 31 kD surface antigens and belonging to the IgG2a but also to the IgG1 subclasses were able to mediate lysis of the parasite in the presence of human non immune serum. The 22 monoclonal antibodies did not identify antigenic differences between the two independently isolated RH and Wiktor strains.     

Occurrence and genetic characterization of Toxoplasma gondii in naturally infected pigs

The protozoan Toxoplasma gondii is an obligate intracellular parasite that infects a wide range of warm-blooded vertebrates. The data about the occurrence of toxoplasmosis in slaughter pigs in the Slovak Republic are still missing. The aim of our study was to estimate the prevalence of toxoplasmosis in pigs from Slovakia during the period of 2006–2010 by ELISA and PCR methods. In sera of 970 slaughter pigs, 2.16% seropositivity to T. gondii was detected. In tissue samples of seropositive pigs the presence of T. gondii DNA was confirmed. In six monitored Slovak regions the seropositivity varied between 1.11 and 3.48%. The statistically significant differences were recorded between the Ko?ice and Pre?ov region. The seroprevalence of toxoplasmosis in sows (4.26%) was two times higher than that in slaughter pigs (2.06%) (OR = 2.12; 95% CI = 0.48–9.36). Presence of Toxoplasma gondii in tissues of seropositive pig isolates was confirmed by TGR1E and B1 genes and analysis of DNA polymorphism at SAG2 and ROP1 genes revealed the presence of virulent strain of genotype I in 85.7% of infected pigs and an avirulent strain (genotype II) in 14.3% of pigs.     

Toxoplasma gondii antibodies in HIV and non-HIV infected Thai pregnant women

Serological evidence for Toxoplasma gondii infection in Thai pregnant women was investigated. One thousand six hundred and sixty-nine blood specimens were collected from 838 HIV-seropositive and 831 HIV-seronegative pregnant women attending the antenatal-care clinic at Siriraj Hospital, Bangkok, Thailand, during a two-year period. Toxoplasma IgG antibody was detected, using a solid-phase enzyme-linked immunosorbent assay in which the membrane protein p-30 was the predominant antigen. IgG positive sera were subsequently examined for IgM antibody by the capture antibody enzyme immunoassay. The IgG antibody was found in 450 (53.7%) HIV seropositive women and 44 (5.3%) non-HIV infected women, with a statistically significant difference (p < 0.0001). Three of the 450 HIV-seropositive and 2 of the 44 HIV-seronegative sera with IgG antibody were positive for IgM antibody against T. gondii. This result suggested that HIV seropositive pregnant women had a higher risk of Toxoplasma infection with increase exposure to their offspring.     

Standardized quantitative enzyme-linked immunoassay for antibodies to Toxoplasma gondii.

A quantitative and standardized enzyme-linked immunosorbent assay is described which uses lyophilized antigen-coated disks for the detection of human antibodies to Toxoplasma gondii. It does not require serial dilution of the test specimen, and the objective absorbance readings are converted into international units per milliliter traceable to the World Health Organization's reference standard preparation of toxoplasma antibodies. It was shown to be highly specific, reproducible, and sensitive. The reagents were stable, without biohazard, and ready for use. Studies of various parameters in the assay indicated that the test conditions were relatively flexible. The enzyme-linked immunosorbent assay results correlated satisfactorily with the methylene blue dye test, the indirect immunofluorescence test, and the passive hemagglutination test.     

Occurrence of antibodies to Toxoplasma gondii in water buffaloes and meat cattle in Rio Grande do Sul State, southern Brazil

Serum samples from 169 water buffaloes and 121 beef cattle were analyzed for antibodies to T. gondii by an indirect fluorescent antibody test (IFAT). Positive results were obtained in 27.2% of water buffaloes and 17.4% of cattle. Statistical analysis indicated significant differences between the prevalence in cattle and buffalo (p ≤ 0.05). The highest titres found in positive animals were 1:256 (buffaloes) and 1:64 (cattle). In both bovine species, toxoplasmosis frequency in young animals (less than 2 years old) was lower compared to older individuals, although the differences seen in cattle were not statistically significant.     

High seroprevalence of antibodies to Toxoplasma gondii in wild animals from Portugal

We report an investigation of antibodies to Toxoplasma gondii in 52 wild birds and 20 wild mammals from northern and central areas of Portugal by using the modified agglutination test. The birds comprised 26 common buzzards (Buteo buteo), five tawny owls (Strix aluco), four white storks (Ceconia ceconia), three Eurasian eagle owls (Bubo bubo), three northern goshawks (Accipiter gentilis), two booted eagles (Hieraaetus pennatus), two common barn owls (Tyto alba), two Eurasian sparrowhawks (Accipiter nisus), two short-toed eagles (Circaetus gallicus), one black kite (Milvus migrans), one Griffin vulture (Gyps fulvus), and one peregrine falcon (Falco peregrinus). The mammals were eight wild boars (Sus scrofa), six red foxes (Vulpes vulpes), two common genets (Genetta genetta), two European badgers (Meles meles), one European roe deer (Capreolus capreolus), and one Iberian wolf (Canis lupus signatus). Fifty percent of the wild birds and 90% of the wild mammals were seropositive; the overall seroprevalence of infection was 61.1%. When comparing the prevalence of antibodies in birds and mammals from northern Portugal, a significant difference was found, but the same was not true for birds and mammals from central Portugal. Seroprevalence levels were 30.0% in juvenile and 62.5% in adult birds (p = 0.046), 0.0% in juvenile and 94.7% in adult mammals (p = 0.100), 80.0% in female and 66.7% in male birds (p = 1.000), and 81.8% in female and 100% in male mammals (p = 0.479). This is the first study performed on T. gondii in birds of prey, white storks, and wild carnivores in Portugal.     

Serological prevalence of Toxoplasma gondii antibodies in pregnant women from Southern Brazil

During 15 months (01 April 2003–31 July 2004), 20,389 women showing positive pregnancy tests were included in a serological evaluation of toxoplasmosis prevalence using automated immunoenzymatic assays. The women’s serum samples were tested for the presence of IgG and/or IgM antibodies. Overall, 53.03% of the women were positive for IgG and 3.26% were positive for IgM; the analysis used a chi-square adherence test and a significance level of 0.05 (χ ²?=?14,720.35; p?=?0.00). To discriminate between recent and past infection, IgG avidity tests (n?=?166) were carried out, of which 28.3% (n?=?47) presented low avidity. The seroconversion index observed in this study was 0.44%. The seroprevalence results obtained were similar to other serology data found in other regions of Brazil. These data demonstrate the importance of continuous regional and national seroepidemiological inquiries to define public health strategies that can revert and reduce serological prevalence, as described in other countries where toxoplasmosis monitoring is mandatory.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.     

Latex agglutination test for detection of antibodies to Toxoplasma gondii.

A resurgence of interest in Toxoplasma gondii has occurred because this coccidian parasite causes lethal infections in immunologically compromised hosts and is responsible for at least 3,000 congenitally infected infants in the United States annually. Thus, rapid, specific, and inexpensive serologic tests are required for routine screening of patients, especially pregnant women. We have developed a latex agglutination test for antibodies to T. gondii which utilizes covalently coupled T. gondii antigens. When compared with an indirect immunofluorescence assay, the latex test had a sensitivity of 94% and specificity of 100%. Compared with an enzyme-linked immunosorbent assay, the latex test had 86% sensitivity and 100% specificity. When testing samples which exhibited nonspecific polar staining by the immunofluorescence assay, the enzyme-linked immunosorbent assay had a 50% false-positive rate, whereas the latex agglutination test yielded no false-positive results. Thus, the latex agglutination test provided an efficacious method for routine serological screening for antibodies to T. gondii.     

The production of hybrid cells producing monoclonal antibodies against Toxoplasma gondii

Hybridomas that secrete monoclonal antibody to Toxoplasma gondii have been developed. Two groups of 10 female BALB/c mice each were immunized either over a shorter (71 d) or longer (117 d) period at first with Toxoplasma lysate antigen and afterwards with intact tachyzoites of the RH strain. Higher titres of antibody were obtained with the long-period immunization. The fusion experiments have shown that both schemes of immunization approximately result in the same number (16 and 14% respectively) of hybridoma cell lines producing antigen-specific monoclonal antibodies. Hybridoma cultures secreting antitoxoplasma monoclonal antibodies were screened parallel by indirect immunofluorescence antibody test (IFAT) and enzyme immunoassay (EIA). 16 of the hybridoma cell cultures produced positive results only in the IFAT, 112 reacted only in the EIA and 21 were positive in both tests. The monoclonal antibodies 5B10, 5G6 and 1B2, which are positive in the IFAT form a chemical compound with the major antigens on the surface of RH tachyzoites. The patterns of fluorescence produced by these monoclonal antibodies are in conformity with those produced by using polyclonal sera of Toxoplasma gondii infected hosts (mouse, rabbit, man).     

Autophagy activated by Toxoplasma gondii infection in turn facilitates Toxoplasma gondii proliferation

Autophagy was found to play an antimicrobial or antiparasitic role in the activation of host cells to defend against intracellular pathogens, at the same time, pathogens could compete with host cell and take advantage of autophagy to provide access for its proliferation, but there are few articles for studying the outcome of this competition between host cell and pathogens. Therefore, the aim of our study was to investigate the relationship between autophagy activated by Toxoplasma gondii (T. gondii) and proliferation of T. gondii affected by autophagy in vitro. Firstly, human embryonic fibroblasts (HEF) cells were infected with T. gondii for different times. The monodansylcadaverine (MDC) staining, acridine orange (AO) staining, punctuate GFP-LC3 distribution, and transmission electron microscopy (TEM) assays were conducted, and the results were consistent in showing that gondii infection could induce autophagy. Secondly, HEF cells were infected with T. gondii and treated with autophagy inhibitor bafilomycin A1 or inducer lithium chloride for different times. Giemsa staining was conducted, and the results exhibited that T. gondii infection-induced autophagy could in turn promote T. gondii proliferation. Simultaneously, the results of Giemsa staining also revealed that autophagy inhibitor could reduce the number of each cell infected with T. gondii and inhibit T. gondii proliferation. In contrast, autophagy inducer could increase the number of each cell infected with T. gondii and encourage T. gondii proliferation. Therefore, our study suggests that T. gondii infection could activate autophagy, and this autophagy could in turn facilitate T. gondii proliferation in HEF cells for limiting nutrients.     

Quantitation of antibodies to Toxoplasma gondii in swine sera by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of antibodies to Toxoplasma gondii in swine sera. Because a commercial anti-swine IgG conjugate was directed also against swine IgM, the conjugate was absorbed with the IgM fraction to eliminate the interference of naturally occurring IgM antibodies that appeared consistently in sera collected from slaughtered pigs at an abattoir. The ELISA values of 0.2 or more observed in most of the sera successfully decreased to less than 0.2 by the use of absorbed conjugate. An attempt to use a protein A conjugate has failed. Evaluation of this system by comparing it with the latex agglutination test provided a high significant correlation, indicating its usefulness for serodiagnosis of swine toxoplasmosis.     

Immunology of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite. Following oral infection the parasite crosses the intestinal epithelial barrier to disseminate throughout the body and establish latent infection in central nervous tissues. The clinical presentation ranges from asymptomatic to severe neurological disorders in immunocompromised individuals. Since the clinical presentation is diverse and depends, among other factors, on the immune status of the host, in the present review, we introduce parasitological, epidemiological, clinical, and molecular biological aspects of infection with T. gondii to set the stage for an in-depth discussion of host immune responses. Since immune responses in humans have not been investigated in detail the present review is exclusively referring to immune responses in experimental models of infection. Systemic and local immune responses in different models of infection are discussed, and a separate chapter introduces commonly used animal models of infection.     

Dubious results of determining the presence of anti-HIV-1 antibodies in blood donors]

In 588 sera of blood donors the Western Blot test (WB) was done for confirming the presence of anti-HIV-1 antibodies. Negative results were obtained in 247 sera and positive in 90. In 251 cases the result was doubtful (42.7%). These results were obtained in 153 cases, however only 104 subjects came for control tests and had from 1 to 5 tests. Eight subjects were returned to the register of blood donors after obtaining of two negative WB results. In 5 cases in control test the serum reacted with the protein of HIV-1, and they were regarded as being carriers. In the remaining 91 subjects doubtful results persisted for from 3 to 12 months. The reactivity of the doubtful sera changed. Most of them reacted with the proteins opceded by one of three genes, they were mostly core proteins, among them p24 protein. The remaining sera reacted with proteins encoded by genes of the envelope and core. The possible causes of these doubtful results are discussed.     

Neospora spp. and Toxoplasma gondii antibodies in horses in the Czech Republic

During January 2007, blood samples were collected from 552 healthy horses from nine different regions of the Czech Republic. Sera were tested for serum antibodies to Neospora caninum by a competitive-inhibition enzyme-linked immunosorbent assay and confirmed by an indirect fluorescent antibody test. The same samples were tested for serum antibodies against Toxoplasma gondii by a latex agglutination test. In total, 131 of 552 (24%) horses reacted positively for Neospora antibodies in competitive-inhibition enzyme-linked immunosorbent assay; seven of them had ≥50% of inhibition. Samples were confirmed in indirect fluorescence test, and only two samples were positive with final titres 50 and 100, while others were negative. Antibodies against T. gondii were found in 125 (23%) horses. This is the first serologic survey for Neospora spp. antibodies performed on horses in the Czech Republic.     

Prevalence of cytomegalovirus antibodies in Thai blood donors

Antibodies to cytomegalovirus (CMV) were determined in Thai blood donors using the complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA). A total of 203 voluntary blood donors, 181 males and 22 females, who came to the Blood Bank at Siriraj Hospital during February 1985, were investigated. Their ages ranged from 17 to 53 years (mean 24.3 +/- 6.9). Seventy-three out of 156 (46.8%) and 171 out of 203 (84.2%) sera were positive for CMV antibodies as detected by the CF test and ELISA respectively. The result of ELISA showed that 95.5 per cent of the female blood donors and 82.9 per cent of the males possessed CMV antibodies. No difference in the geometric mean titres of either sex was noted. The findings indicated that ELISA was more sensitive than the CF test for detecting CMV antibodies. The high percentage of CMV-seropositive blood donors indicates that CMV infection is common in this country. Therefore, it might be necessary to test blood donors for CMV antibodies when they are giving blood for use by certain patients, especially immunocompromised ones; the same observation applies with regard to organ donors before transplantation is carried out.