Toxoplasma Antigen Research Articles

Heterologous immunization targeting the CST1 antigen confers better protection than ROP18 in mice.

Aim: To evaluate the protective efficacy induced by heterologous immunization with recombinant baculoviruses or virus-like particles targeting the CST1 and ROP18 antigens of Toxoplasma gondii.Materials & methods: Recombinant baculovirus and virus-like particle vaccines expressing T. gondii CST1 or ROP18 antigens were developed to evaluate protective immunity in mice upon challenge infection with 450 Toxoplasma gondii (ME49).Results: Immunization with CST1 or ROP18 vaccines induced similar levels of T. gondii-specific IgG and IgA responses. Compared with ROP 18, CST1 vaccine showed better antibody-secreting cell response, germinal center B cell activation, and significantly reduced brain cyst burden and body weight loss.Conclusion: Our findings suggest that CST1 heterologous immunization elicited better protection than ROP18, providing important insight into improving the toxoplasmosis vaccine design strategy.

Role of <i>Toxoplasma gondii</i> in thyroiditis in pregnant women

Toxoplasmosis (acute and latent) is the most prevalent parasitic infection worldwide and can be associated with some problems in pregnant women. Thyroid diseases are the most common endocrine disorders secondary to diabetes among pregnant women. Previous studies proposed a relationship between latent еoxoplasmosis (LT) and autoimmune thyroiditis diseases (AITDs). This study intended to investigate the frequency and correlation between toxoplasmosis and AITD in pregnant women. In this cross-sectional study, the statistical population included 1248 pregnant women at the gestational age of 916 weeks and in Tehran. The Toxoplasma IgM and IgG tests were assessed with enzyme-linked immunosorbent assay (ELISA). The diagnostic criteria for toxoplasmosis were abnormal IgG and IgM titers. In addition, FT4, TPO Ab, and TSH were evaluated using enzyme-linked fluorescence immunoassay (ELFA). TPO Ab was used to distinguish thyroid patients with autoimmune origin from those with other thyroiditis diseases. The analysis showed no significant relationship between keeping a house cat and acute toxoplasmosis. Acute and latent toxoplasmosis represented 3.4% and 29.6%, respectively. The frequency of thyroid diseases was 18.8% (hypothyroidism 15.8% versus hyperthyroidism 3%). The frequency of autoimmune thyroiditis diseases (AITDs) was 5.5%, and 27.9% of subjects with latent toxoplasmosis (LT) had a thyroiditis disease, but 13.8% of pregnant women with LT had only AITD with a significant correlation (p 0.001). Results show that Toxoplasma IgG+ can increase the risk of AITD by 10.39-fold and a higher TPO Ab titer in people with LT. It seems Toxoplasma gondii may cause thyroiditis in pregnant women likely because antigenic similarity of Toxoplasma and thyroperoxidase leads to cross-reactivity in the immune system, potentially causing AITD. It might be said that the high prevalence of LT among pregnant women may have a potential role in the stimulation of the immune system to the development of autoimmune diseases, such as AITD. So future studies could be conducted with a focus on discovering molecular similarities between thyroperoxidase and Toxoplasma antigens.

Assessing seroprevalence and associated risk factors for multiple infectious diseases in Sabah, Malaysia using serological multiplex bead assays.

Infectious diseases continue to burden populations in Malaysia, especially among rural communities where resources are limited and access to health care is difficult. Current epidemiological trends of several neglected tropical diseases in these populations are at present absent due to the lack of habitual and efficient surveillance. To date, various studies have explored the utility of serological multiplex beads to monitor numerous diseases simultaneously. We therefore applied this platform to assess population level exposure to six infectious diseases in Sabah, Malaysia. Furthermore, we concurrently investigated demographic and spatial risk factors that may be associated with exposure for each disease. This study was conducted in four districts of Northern Sabah in Malaysian Borneo, using an environmentally stratified, population-based cross-sectional serological survey targeted to determine risk factors for malaria. Samples were collected between September to December 2015, from 919 villages totaling 10,100 persons. IgG responses to twelve antigens of six diseases (lymphatic filariasis- Bm33, Bm14, BmR1, Wb123; strongyloides- NIE; toxoplasmosis-SAG2A; yaws- Rp17 and TmpA; trachoma- Pgp3, Ct694; and giardiasis- VSP3, VSP5) were measured using serological multiplex bead assays. Eight demographic risk factors and twelve environmental covariates were included in this study to better understand transmission in this community. Seroprevalence of LF antigens included Bm33 (10.9%), Bm14+ BmR1 (3.5%), and Wb123 (1.7%). Seroprevalence of Strongyloides antigen NIE was 16.8%, for Toxoplasma antigen SAG2A was 29.9%, and Giardia antigens GVSP3 + GVSP5 was 23.2%. Seroprevalence estimates for yaws Rp17 was 4.91%, for TmpA was 4.81%, and for combined seropositivity to both antigens was 1.2%. Seroprevalence estimates for trachoma Pgp3 + Ct694 were 4.5%. Age was a significant risk factors consistent among all antigens assessed, while other risk factors varied among the different antigens. Spatial heterogeneity of seroprevalence was observed more prominently in lymphatic filariasis and toxoplasmosis. Multiplex bead assays can be used to assess serological responses to numerous pathogens simultaneously to support infectious disease surveillance in rural communities, especially where prevalences estimates are lacking for neglected tropical diseases. Demographic and spatial data collected alongside serosurveys can prove useful in identifying risk factors associated with exposure and geographic distribution of transmission.

Evaluation of cytotoxic activity of live toxoplasma gondii tachyzoites and toxoplasma antigen on MCF-7 human breast cancer cell line

The aim of this study was to investigate the cytotoxic potency of live Toxoplasma gondii tachyzoites as well as Toxoplasma antigen on MCF-7 human breast cancer cell line. Cancer cell lines are considered an essential preliminary step towards in-vitro investigation of the potential antineoplastic impact of novel chemotherapeutic agents. Pathogens, including viruses, bacteria, and parasites are noticeably under investigation, considering their potential antineoplastic activity. Some have attained a steady position in the clinical field as hepatitis B virus, human papilloma virus and BCG immunization. Toxoplasma gondii is an apicomplexan parasite with promising antineoplastic activity. In this study, live Toxoplasma tachyzoites provoked a direct cytotoxic effect on MCF-7 in a dose dependent manner, while Toxoplasma antigen didn’t induce such impact.
 Skipping the direct cytotoxic effect of Toxoplasma antigen doesn’t totally divert the possible antineoplastic activity of Toxoplasma antigen. Potential alternative immune mediated mechanisms could be an alternative. Further in-vivo studies in different cancer models are mandatory to investigate the underlying mechanisms of antineoplastic activity of Toxoplasma gondii

Prevalence of toxoplasmosis in semi-domesticated and pet cats within and around Bangkok, Thailand

BackgroundToxoplasmosis is one of the most common parasitic zoonoses worldwide. Cats become infected after ingesting infected tissue cysts. The objective of the present study was to compare the prevalence of toxoplasmosis in pet cats and semi-domesticated cats in the Bangkok metropolitan region. A survey of Toxoplasma infection was conducted in 260 cats (median age [range]: 3 years [10 months–10 years]; 155 females and 105 males) by collecting blood samples from 130 client-owned pet cats and 130 semi-domesticated cats within and around Bangkok during 2016–2017 using indirect fluorescence antibody tests. An IgG antibody to Toxoplasma antigen ratio of ≥1:100 was considered positive for Toxoplasma infection.ResultsThe overall prevalence of T. gondii in cats was 6.5% (17/260). The prevalence of T. gondii in semi-domesticated cats and pet cats was 11.5 and 1.5%, respectively. Semi-domesticated cats aged 1–5 years (14.9%) had a higher prevalence of infection than domesticated cats (1.3%, p = 0.002) of the same age. The odds (95% confidence interval [CI]) of having T. gondii infection in semi-domesticated cats were 8.34 (1.86–76.29, p = 0.0017) times higher than in pet cats. Interestingly, there was an association between T. gondii infection according to city ​region (p = 0.002). The odds (95% CI) of having T. gondii infection in cats living in the inner city were 4.96 (1.03–47.16, p = 0.023) times higher than cats living in the suburb and the vicinity.ConclusionsThe present study identified a higher prevalence of Toxoplasma infection in semi-domesticated cats compared with pet cats. The semi-domesticated cats could serve as a zoonotic reservoir. Public health regulations should be implemented to prevent toxoplasmosis spread.

Multi-epitope vaccine expressed in Leishmania tarentolae confers protective immunity to Toxoplasma gondii in BALB/c mice

Current study deals with a novel multi-epitope vaccine designed in silico and its confirmation experiments for potential efficacy in BALB/c mice. Major histocompatibility complex (MHC)-binding and B-cell binding epitopes of five Toxoplasma antigens (SAG1, ROP16, GRA12, MIC4 and M2AP) were predicted. Selected epitopes were fused together using SAPGTP linker, and antigenicity, allergenicity, physico-chemical features, secondary and tertiary structures and validations were all performed via bioinformatics servers. Then, vaccine construct was cloned into pLEXSY-neo 2.1 vector. After Leishmania tarentolae transfection, live recombinant and wild parasites were subcutaneously injected into 6–8 week female BALB/c mice and immune responses were measured. Results showed that the peptide possessed 282 amino acid residues with average molecular weight of 28.06 kDa. About 90% of the peptide residues were incorporated in favored and allowed regions of the Ramachandran plot. Vaccinated mice showed remarkably elevated levels of specific antibodies (P < 0.05) with predominance of IgG2a production. Also, a Th1 immune response with production of IFN-γ and relatively increased survival rate against intraperitoneal challenge with RH strain was demonstrated in immunized mice than control groups (P < 0.05). Also, very low levels of IL-4 were demonstrated, which showed statistically significant association with controls (P < 0.05). The findings clarified that multi-epitope vaccine expressed in Leishmania tarentolae induced significant immune responses against acute toxoplasmosis.

Serological Responses to Toxoplasma gondii and Matrix Antigen 1 Predict the Risk of Subsequent Toxoplasmic Encephalitis in People Living With Human Immunodeficiency Virus (HIV).

Clinically useful predictors for fatal toxoplasmosis are lacking. We investigated the value of serological assays for antibodies to whole Toxoplasma antigens and to peptide antigens of the Toxoplasma cyst matrix antigen 1 (MAG1), for predicting incident toxoplasmic encephalitis (TE) in people living with human immunodeficiency virus (HIV; PLWH). We performed a nested case control study, conducted within the Multicenter AIDS Cohort Study (MACS), using serum samples obtained 2 years prior to diagnosis of TE from 28 cases, and 37 HIV disease-matched Toxoplasma seropositive controls at matched time-points. Sera were tested for Toxoplasma antibodies using a commercial assay and for antibodies to MAG1_4.2 and MAG1_5.2 peptides in enzyme-linked immunosorbent assay (ELISA). Two years prior to clinical diagnosis, 68% of TE cases were MAG1_4.2 seropositive compared with 16% of controls (odds ratio [OR] 25.0, 95% confidence interval [CI] 3.14-199.18). Corresponding results for MAG1_5.2 seropositivity were 36% and 14% (OR 3.6, 95% CI .95-13.42). Higher levels of antibody to MAG1_4.2 (OR 18.5 per doubling of the optical density [OD] value, 95% CI 1.41-242) and to Toxoplasma (OR 2.91 for each OD unit increase, 95% CI 1.48-5.72) were also associated with the risk of TE. When seropositivity was defined as the presence of MAG1 antibody or relatively high levels of Toxoplasma antibody, the sensitivity was 89% and specificity was 68% for subsequent TE. Antibodies to MAG1 showed predictive value on the occurrence of TE in PLWH, and the predictive performance was further improved by adding the levels of Toxoplasma antibody. These measures could be clinically useful for predicting subsequent diseases in multiple at-risk populations.

Serotyping, host genes and cytokines response in human ocular toxoplasmosis

In human ocular toxoplasmosis, serotype is related with greater severity. We analyzed Toxoplasma GRA6 serotype in 23 patients with ocular toxoplasmosis (13 confirmed, two co-infections- and eight unconfirmed cases) and 20 individuals chronically infected with Toxoplasma but without ocular involvement. In patients with ocular toxoplasmosis, we also studied host gene polymorphisms related to immune response (IL-1β; IL-1α; IL-10; IFN-γ; TNF-α, IL-12), IL-17R, TLR-9, and P2RX7. Additionally, eight patients were studied for the production of TNFα, IL1-β, IFN−γ and IL-10 by their peripheral leukocytes after ex vivo stimulation with soluble Toxoplasma antigens. There were no differences in the distribution of serotypes (GRA6-I versus GRA6 non-I) between infected individuals with- or without ocular involvement. Seropositivity for GRA6-I was associated with higher number of retinal lesions and higher levels of IL-1β. Two polymorphisms were associated with specific clinical manifestations of ocular toxoplasmosis: IL-10 -819 C/T with bilateral lesions and IL-12 + 169,774 A/C with synechia. Higher levels of IL-10 were found in patients with the allele G/G at the polymorphic region IL-10 -1082. People with a GRA6 I serotype and possessing the allele G/G at the polymorphic region TNFα-857 suffered from an increased number of retinal lesions. We found a positive association between host cytokine genes polymorphisms and GRA6 serotypes correlated with specific clinical manifestations and immune response in ocular toxoplasmosis.

Intrinsic Impairment of CD8 T Cells Induced by Helminth Coinfection

Abstract Although it is known that helminth-triggered type 2 response increases host vulnerability to coinfections and potentially decreases vaccine efficacy, the exact mechanism(s) for the helminth-induced immunomodulation is still a fundamental question in the field. We have previously reported that CD44hiCD62L−KLRG1+ effector CD8 T cells from mice coinfected with Heligmosomoides polygyrus (H. polygyrus) and Toxoplasma gondii (T. gondii) exhibit a suppressed effector differentiation and impaired IFNγ production upon in vitro re-stimulation with toxoplasma antigens. This impairment was accompanied by a decrease in both, mitochondrial biomass and gene expression of the mitochondrial transcription factor, TFB1M. Further analysis of CD44hiT. gondii-specific CD8 T cells via single cell RNAseq, revealed an effector CD8 T cell subset from coinfected mouse that exhibit down-regulation of Cox17 and Atp5k transcripts. Both of these transcripts encode for proteins that belong to the electron transport chain multimeric complexes III and V, respectively. Taken altogether, our data suggest a potential metabolic-dependent mechanism of effector CD8 T cell insufficiency induced by helminth-coinfection. We hypothesize that helminth infection results in an impairment of CD8 T cell metabolic reprogramming, thus, affecting their effector development and function in response to T. gondii infection. The aim of this work is to increase the understanding of the helminth-associated immunomodulatory mechanism of CD8 T cells with the objective of finding therapeutic ways to restore CD8 T cells effector functions to improve vaccine efficacy in helminth-affected subpopulations.

Serological evidence of Toxoplasma gondii infection as potential risk for the development of lepromatous leprosy in an endemic area for both neglected tropical diseases in Brazil

BackgroundMycobacterium leprae and Toxoplasma gondii infections are both neglected tropical diseases highly prevalent in Brazil. Infection with certain parasite species can significantly alter susceptibility to other important pathogens, and/or influence the development of pathology. Here we investigated the possible influence of M. leprae/T. gondii co-parasitism on the manifestation of leprosy and its clinical forms.MethodsParticipants (n = 291) were recruited in Campos dos Goytacazes city, Rio de Janeiro state, southeast Brazil, from August 2015 to December 2019 and clinically diagnosed for leprosy. Participants were selected based on the presence (patients) or absence (healthy controls) of the leprosy disease. Contacts of patients were also recruited for this study. Serum samples from patients (n = 199) with leprosy, contacts (n = 40) and healthy controls (n = 52) were investigated for levels of IgM and IgG anti-phenolic glycolipid-1 (PGL-1) by ELISA. Additionally, IgG antibody against soluble Toxoplasma antigen (STAg) was measured in sera samples from leprosy patients, contacts and healthy controls for Toxoplasma gondii serology by ELISA. Anti-PGL-1 IgG and IgM levels were compared using one-way ANOVA Kruskal-Wallis or Mann-Whitney, while Spearman test was used to correlate levels of IgG anti-STAg and IgM/IgG anti-PGL-1 from seropositive and seronegative individuals for T. gondii infection. The risk of T. gondii infection for leprosy disease was assessed using Fisher’s test.ResultsLevels of IgM anti-PGL-1 antibodies were significantly higher in multibacillary (MB) patients compared to paucibacillary (PB) patients (P = 0.0068). Higher IgM and IgG levels anti-PGL-1 were detected in patients with the lepromatous forms. The serologic prevalence for T. gondii infection was 74.9%. We detected increased anti-STAg antibody levels in leprosy patients (79.4%), reaching 88.8% within those with lepromatous form of this disease. The leprosy risk increase in T. gondii seropositive individuals was two-fold (odds ratio [OR] = 2.055; 95% confidence intervals [95% CI]: 1.18–3.51) higher than those seronegative, and considering the lepromatous leprosy risk this increase was even dramatic (OR = 4.33; 95% CI: 1.76–9.69) in T. gondii seropositive individuals. Moreover the leprosy risk in T. gondii seropositive individuals was weakly correlated to the levels of IgG anti-STAg and IgM/IgG anti-PGL-1.ConclusionsAltogether, our results suggest that T. gondii infection may exert immunomodulatory properties that influence to the susceptibility of leprosy, mainly on its more severe clinical form. A better understanding of parasite immunomodulation can ultimately contribute to the development of medical applications.

Radiation effects on Toxoplasma antigens: different immune responses of irradiated intact tachyzoites or soluble antigens in experimental mice models

Purpose: Purpose: Protein irradiation causes aggregation, chain breakage, and oxidation, enhancing its uptake by antigen-presenting cells. To evaluate if irradiated proteins participate on the protection, we studied the immune response induced in mice immunized with irradiated soluble extracts of T. gondii tachyzoites (STag) or irradiated intact T. gondii RH tachyzoites (RH0.25 kGy).Material and Methods: Soluble extracts of Toxoplasma gondii tachyzoites (STag) were irradiated at different dose by Cobalt-60 source. By polyacrylamide gel electrophoresis (SDS-Page) we evaluated the effects on primary structures of protein STags induced by irradiation. By Enzyme-linked Immunosorbent Assay (ELISA) we evaluated the difference between humoral immune response induced by irradiated STag or RH tachyzoites in immunized mice from the detection of specific immunoglobulin G (IgG) antibodies in the serum of immunized mice. From challenge with viable RH strain of T. gondii we evaluated the protection induced in the immunized animals. By cytometry we performed the phenotyping of T and B lymphocytes in the peripheral blood of the immunized animals.Results: Irradiation dose of 1.5 kGy induced minimal changes in most proteins, without affecting their antigenicity or immunogenicity. Immunization showed saturation at the dose of 10 µg/mice, with worst response at higher doses. STag irradiated at 1.5 kGy (STag1.5 kGy) induced higher survival and protection similar to T. gondii RH strain irradiated at 0.25 kGy (RH0.25 kGy), with higher serum levels of high affinity IgG compared to STag native. Blood immune memory cells of mice immunized with STag1.5 kGy had higher proportions of CD19+ (cluster of differentiation 19) and CD4+ (cluster of differentiation 14) cells, whereas mice RH0.25 kGy had high proportion of memory CD8+ (cluster of differentiation 8) cells.Conclusions: Our data suggest that major histocompatibility complex type I (MHCI) pathway, appears seem to be used by RH0.25 kGy to generate cytotoxic cells while STag1.5 kGy uses a major histocompatibility complex type II (MHCII) pathway for B-cell memory, but both induce sufficient immune response for protection in mice without any adjuvant. Irradiation of soluble protein extracts enhances their immune response, allowing similar protection against T. gondii in mice as compared to irradiated intact parasites.

The first study on the usefulness of recombinant tetravalent chimeric proteins containing fragments of SAG2, GRA1, ROP1 and AMA1 antigens in the detection of specific anti-Toxoplasma gondii antibodies in mouse and human sera.

This study presents an evaluation of four tetravalent recombinant chimeric proteins containing fragments of the Toxoplasma gondii antigens, SAG2, GRA1, ROP1 and AMA1, as potential replacements of a the soluble, whole-cell tachyzoite lysate (TLA) used in serological assays. Recombinant chimeric proteins (SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1) obtained by genetic engineering were tested for their reactivity with specific IgM and IgG antibodies from sera of experimentally infected mice and humans with T. gondii infection using an enzyme-linked immunosorbent assay (ELISA). In total 192 serum samples from patients with acquired T. gondii infection and 137 sera from seronegative individuals were examined. The reactivity of chimeric antigens with antibodies generated during T. gondii invasion was measured and compared to the results obtained in assays based on whole-cell Toxoplasma antigen. Chimeric proteins proved effective in differentiation between T. gondii-infected and uninfected individuals (100% sensitivity and specificity in the IgG ELISAs) which shows their potential usefulness as a replacements for TLA in standardized commercial tests for the serodiagnosis of toxoplasmosis. In addition, the chimeric proteins were tested for use in avidity determination. Obtained results were comparable to those of the corresponding commercial assays, suggesting the utility of these proteins for avidity assessment. Furthermore, this study demonstrated that the AMA1-SAG2-GRA1-ROP1 chimeric protein has the potential to distinguish specific antibodies from serum samples of individuals with the early and chronic phase of T. gondii infection.

Determination the Time of Toxoplasmosis among Pregnant Women by using IgG Avidity to Various Toxoplasma Gondii Antigens

Toxoplasmosis is caused by Toxoplasma gondii, which is an intracellular protozoan parasite. Its main host is the cat. It is one of the most common human parasites. The main objective of this study is to determine the time of T. gondii infection among pregnant women by using avidity test of Toxoplasma IgG to various Toxoplasma antigens. The study period was from August 2017 to February 2018 to study the time of toxoplasmosis among 180 pregnant women and 100 non pregnant married women as control attending to hospitals, primary health care centers and some private medical laboratories. The pregnant women were examined for Toxo-IgG seroprevalence by using ELISA technique, and then examined their avidity of antibodies for specific Toxoplasma antigens by using line immune assay. The rates of Toxo-IgG seropositive were64 (35.56 %) among pregnant women. Regarding the reactivity of determined Toxo-IgG against various Toxoplasma antigens, the rates were 40(62.50%), 39 (60.93%), 63(98.43%) ,55(85.93%) ,63(98.43%), 62(96.87%) ,41(64.04%) and 42(65.62) positive for Toxoplasma ROP1C, MIC3, GRA7, GRA8, p30, MAG1,GRA1,rSAG1 antigens, respectively. Considering the avidity of Toxo-IgG for these antigens, the rates of high avidity were higher than low and intermediate avidity. So, the highest rate of high avidity was 85.0% for ROP1c antigen. It is concluded that the highest rate of predicting Toxoplasma infection among pregnant women was for a period of more than 6 months, which makes it less dangerous for maternal and fetal health..

Seroprevalence of Toxoplasma-IgA and its Relation to Serum IL-4 Level Among Pregnant Women.

Toxoplasmosis is caused by infection with the parasite Toxoplasma gondii. It is one of the most common parasitic infections in humans and is most typically asymptomatic. However infection in a pregnant woman can cause severe and disabling disease in the developing fetus. The presented study aimed to determination the correlation of Toxo-IgA seroprevalence among pregnant women to various Toxoplasma antigens with the serum IL-4 level. From the June 2017 to January 2018, 300 pregnant women enrolled in this study attended to hospitals, primary health care centers and some private medical laboratories in Kirkuk. The pregnant women were examined for Toxo-IgA and determine their specificity for various Toxoplasma antigens by using line immune assay and the serum IL-4 evaluated by using ELISA technique. The rates of Toxo-IgA seropositive were 22 (7.33 %). Considering the reactivity of determined Toxo-IgA against various Toxoplasma antigens; the rates were 12(54.54%), 13(59.09%), 11(50.00%), 14(63.63%), 17(77.27%), 13(59.09%), 11(50.00%) and 18(81.81%) seropositive for Toxoplasma ROP1C, MIC3, GRA7, GRA8, p30, MAG1, GRA1 and rSAG1 antigens respectively. Regarding to the total serum IL-4 level, the highest rate of decreased serum IL-4 level among Toxo-IgA seropositive was 59.09%.In the presented study we concluded the highest rates of decreased serum IL-4 levels was 54.55% seen within Toxo-IgA seropositive for GRA7 and GRA1 antigens , and the rates of increased serum IL-4 level ranged from 5.88% to 9.09% within all seropositive groups to various Toxoplasma antigens.

Evaluation of LIPS (luciferase immunoprecipitation system) for serodiagnosis of Toxoplasmosis

Development of reliable, quantitative technologies for serodiagnosis of Toxoplasma gondii infection remains desirable. The luciferase immunoprecipitation system (LIPS) is a relatively simple, highly sensitive, and rapid quantitative immunoassay. The major advantages of this assay over ELISA are a wider dynamic range, shorter overall assay time, and less sample volume. In this study, we aimed to use this method for the serodiagnosis of toxoplasmosis. Recombinant Toxoplasma antigens (dense granule antigens GRA6, GRA7, and GRA8 and bradyzoite antigen BAG1) fused with nanoluciferase (Nluc, a small luciferase enzyme) were expressed in Escherichia coli, purified, and tested in LIPS assays with sera from experimental mice infected with T. gondii and a WHO standard anti-Toxoplasma human immunoglobulin (TOXM). In the experimentally infected mice, LIPS assays detected antibodies against Nluc-GRA6, Nluc-GRA7, and Nluc-GRA8 as early as day 14, whereas antibodies against Nluc-BAG1 remained undetected until day 21 and then showed significant elevation on day 60. In TOXM sera, LIPS assays with each Nluc recombinant protein produced reliable standard curves with a coefficient of determination (R2) of 0.980–0.989 for GRA6, 0.986–0.990 for GRA7, 0.998–0.999 for GRA8, and 0.942–0.987 for BAG1. The detection limits were estimated to be 3.9, 2, 1, and 1 IU/ml for rGRA6, rGRA7, rGRA8, and rBAG1, respectively. The LIPS assay for toxoplasmosis could detect antibodies against T. gondii in the mouse and human sera with a reasonably high sensitivity. We consider the LIPS assay to be a promising alternative tool for screening, diagnosing, and monitoring toxoplasmosis. In particular, detection of antibodies against BAG1 may be useful for a longitudinal seroprevalence study in suspected high-risk areas on the basis of its elevated serum concentration in the chronic phase.

Construction and Sequencing of Dense Granular14 (GRA14) Gene of Toxoplasma gondii (RH) in Expression Prokaryotic Plasmid PET32a: A Preliminary Study in Vaccine Production

Introduction: Toxoplasma gondii is an obligatory intracellular protozoan parasite, which infects human beings. Since the current antigens used for diagnosis or vaccination are contaminated with non -parasitic material in which the parasite is grown, it is tried to produce recombinant antigens to design vaccines against toxoplasmosis, or make diagnostic kits. Choosing the type of antigen to produce recombinant vaccine or diagnostic kits is considerably important. The dense granule protein 14 (GRA14) gene is one of the excretory-secretary antigens of Toxoplasma which seems to be an appropriate candidate in production of recombinant vaccines and diagnostic kits. The current study aimed to clone GRA14 gene of T. gondii (RH) in a cloning vector for further production of dense granular proteins. Materials and Methods: Genomic DNA was isolated from tachyzoite of parasite by phenol chloroform method and gene fragment was amplified by polymerase chain reaction (PCR). The PCR products were ligated into restriction enzymes sites of pTG19-T cloning vector. Then transformed into Escherichia coli Top10 strain and screened by IPTG and X-Gal. Then recombinant plasmid confirmed by the colony-PCR and restriction enzyme digestion using SacI and NotI was done followed by sequencing .After isolation of this gene from pTG19-T, it was subcloned into a prokaryotic expression plasmid (pET32a). The pET32a - GRA14 constructs were analyzed by PCR, restriction analysis and sequencing. Results: Evaluation of PCR products by agarose gel electrophoresis and analysis of nucleotide sequencing of 1227 bp gene encoding the protein GRA14, revealed the complete homology with the recorded sequences in the gene bank. After enzyme restriction and electrophoresis a fragment about 1227 bp was separated from pET32a. Conclusions: The result of this study showed that recombinant GRA14 Toxoplasma was constructed successfully and ready for future study which seems like the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit.

Advantages of bioconjugated silica-coated nanoparticles as an innovative diagnosis for human toxoplasmosis

Nanotechnology is a promising arena for generating new applications in Medicine. To successfully functionalised nanoparticles for a given biomedical application, a wide range of chemical, physical and biological factors have to be taken into account. Silica-coated nanoparticles, (SiO2NP) exhibit substantial diagnostic activity owing to their large surface to volume ratios and crystallographic surface structure. This work aimed to evaluate the advantage of bioconjugation of SiO2NP with PAb against Toxoplasma lyzate antigen (TLA) as an innovative diagnostic method for human toxoplasmosis. This cross-sectional study included 120 individuals, divided into Group I: 70 patients suspected for Toxoplasma gondii based on the presence of clinical manifestation. Group II: 30 patients harboring other parasites than T. gondii Group III: 20 apparently healthy individuals free from toxoplasmosis and other parasitic infections served as negative control. Detection of circulating Toxoplasma antigen was performed by Sandwich ELISA and Nano-sandwich ELISA on sera and pooled urine of human samples. Using Sandwich ELISA, 10 out of 70 suspected Toxoplasma-infected human serum samples showed false negative and 8 out of 30 of other parasites groups were false positive giving 85.7% sensitivity and 84.0% specificity, while the sensitivity and specificity were 78.6% and 70% respectively in urine samples. Using Nano-Sandwich ELISA, 7 out of 70 suspected Toxoplasma-infected human samples showed false negative results and the sensitivity of the assay was 90.0%, while 4 out of 30 of other parasites groups were false positive giving 92.0% specificity, while the sensitivity and specificity were 82.6% and 80% respectively in urine samples. In conclusion, our data demonstrated that loading SiO2 nanoparticles with pAb increased the sensitivity and specificity of Nano-sandwich ELISA for detection of T.gondii antigens in serum and urine samples, thus active (early) and light infections could be easily detected.

Detection of Toxoplasma gondii in cat's internal organs by immunohistochemistry methods labeled with-[strept] avidin-biotin.

Aim:The aims of the study are to detect the presence of Toxoplasma gondii antigen and to determine its distribution location in several organs of domestic cat using immunohistochemistry (IHC) method with Labeled-[Strept] Avidin-Biotin (LAB-SA).Material and Methods:Four domestic cats aged 1-2 years were used as sample in this research. The sample divided into two groups with two cats each. Cats in Group I were positive Toxoplasma based on serologically screening test, while cats in Group II were orally infected with 1×106 Toxoplasma oocyst. All samples then necropsied, and the organs including brain, liver, kidney, duodenum, jejunum, ileum, lungs, and spleen were collected for IHC method with LAB-SA.Result:The result showed that Toxoplasma antigens were detected in ileum of both serologically positive domestic cat and the experimentally infected cats. Toxoplasma was also observed in kidney of serologically positive domestic cat. In the serologically positive domestic cat, necrotic lesions were found on ileum, kidney, and liver, whereas in experimentally infected cat, the lesion was only found on ileum.Conclusion:The presence of Toxoplasma antigen is successfully detected in several organs of domestic cat using IHC method with the LAB-SA.

Toxoplasmosis, leptospirosis, and brucellosis seroepidemiology in veterinary medical students and their relation with unique health

Toxoplasmosis, leptospirosis, and brucellosis are global zoonoses, with humans as accidental participants in their transmission cycles. The can also be considered occupational diseases, because certain professionals are at greater risk of contact and infection by such zoonoses. These three diseases have different epidemiological characteristics because of the distinct environmental, social, cultural, and economic conditions where these pathogens circulate. Because of the importance of these diseases and their associations with specific occupations, we performed a seroepidemiological survey of Toxoplasma, Leptospira, and Brucella antibodies, with an analysis of the association between positive serum and certain occupational and environmental variables, in students of a veterinary medicine course (1st, 2nd, 3rd, 4th, and 5th year) in a university in the northwestern region in the state of Parana, Brazil. From May to November 2014, blood samples were collected from 157 volunteers by professionals trained in nursing and biomedicine from the same university as the veterinary students. At the time of blood collection, the students did not present any clinical signs of the three diseases of interest. To detect anti-Toxoplasma gondii, anti-Leptospira spp., and anti-Brucella antibodies, indirect immunofluorescence test (IIF), microscopic agglutination test (MAT), fast agglutination test (a screening test), and enzyme-linked immunosorbent assay (ELISA) were performed. To determine variables associated with these infections, the students were interviewed to complete an epidemiological questionnaire with environmental, behavioral, and occupational information. The associations between these variables and infections were assess by chi-square or Fischer's exact tests, with a 5% significance level (?). Of the 157 serum samples analyzed, 29.29% reacted to Toxoplasma antigens, with titers ranging from 16 to 4096 by IIF, 1.27% to Leptospira antigens, with titers ranging from 100 to 800 by MAT using Hardjo and Wolffi serovars, and 0.63% to Brucella antigens by ELISA; however, no variables were found to be associated with infection with any of these pathogens. The results of this study show that one-third of the students in the veterinary medicine course were exposed to Toxoplasma gondii, Leptospira spp., and Brucella spp. at some stage in their lives; however, it is not possible to determine whether these infections were acquired at the university, because no associations between occupational risk variables and these infections were found. An understanding of the transmission of each etiological agent and methods to prevent infection is important to maintain low prevalence levels of these zoonotic diseases during the veterinary medicine course and extra-curricular internships, when there is increased exposure to these pathogens.

Toxoplasma gondii: Immunological response of sheep to injections of recombinant SAG1, SAG2, GRA1 proteins coupled to the non-toxic microparticle muramyl dipeptide

BackgroundToxoplasma gondii is an important cause of reproductive loss in sheep and has a large economic impact, worldwide, because of infections in humans and animals. The immune response in sheep was evaluated, following administration with four different combinations of recombinant proteins derived from T. gondii. Methods25 three-year-old non-pregnant Coopworth ewes were vaccinated twice by intramuscular injection with either the recombinant surface antigen 1 (SAG1), 2 (SAG2) or excretory/secretory antigen (GRA1) each individually conjugated to non-toxic muramyl dipeptide (MDP). Blood samples were collected from all animals prior to the first injection and once per week until five weeks after the second injection. Immunoglobulins (Ig) G1 and IgG2 level were measured using an ELISA test. IFN-γ were estimated using the commercially available bovine IFN-gamma test kit (Bovigam, Prionics AG). ResultsAdministration of the recombinant GRA1 enhanced both IFN-γ production from peripheral blood cells when cultured in vitro with Toxoplasma antigen, and GRA1-specific IgG2 antibody level present in serum. Unlike GRA1 SAG1 did not stimulate IFN-γ production in the same test system. ConclusionThese results indicate the potential of recombinant GRA1, as a vaccine candidate to protect sheep against T. gondii infection.