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Development of an immunochromatographic assay based on dense granule protein 7 for serological detection of Toxoplasma gondii infection.

ABSTRACT: Dense granule antigen proteins derived from Toxoplasma gondii (TgGRAs) are potential antigens for the development of diagnostic tools. TgGRA7 and TgGRA14 were detected in the peritoneal fluid of T. gondii-infected mice, suggesting that TgGRAs may be highly antigenic proteins. Here, TgGRA7 and TgGRA14 were evaluated as candidates for the development of a marker for a rapid diagnostic test. The specificity and sensitivity of purified recombinant proteins of TgGRA7 and TgGRA14 were compared in an indirect enzyme-linked immunosorbent assay (iELISA) using a series of serum samples from T. gondii-experimentally infected mice and using recombinant T. gondii major surface antigen 2 (TgSAG2) as a reference control. The iELISA with TgGRA7 showed the greatest diagnostic accuracy and could detect anti-TgGRA7 antibody in acute and chronic infections. A total of 59 field samples from pigs were also examined by the iELISAs, and the results compared with those of the latex agglutination test (LAT). Among the three recombinant antigens, TgGRA7 had the highest rates of positivity, with significant concordance (88.14) and kappa value (0.76) in comparison with the results using LAT. Furthermore, an immunochromatographic test (ICT) based on recombinant TgGRA7 was developed for rapid detection of antibodies to the infection. The ICT differentiated clearly between sera from T. gondii-infected mice and uninfected or Neospora caninum-infected mice. Pig sera were examined with the ICT, and the results compared favorably with those of LAT and iELISA for TgGRA7, with kappa values of 0.66 and 0.70 to 0.79, respectively. These data suggest that the ICT based on TgGRA7 is a promising diagnostic tool for routine testing in the clinic and mass screening of samples in the field.

SUBMITTER: Terkawi MA

PROVIDER: S-EPMC3623415 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Project description:Background and aimToxoplasma gondii is recognized as a zoonosis causing toxoplasmosis in animals globally. Cat is a definitive host of T. gondii and sheds oocyst through feces, which can infect human beings and animals through contaminated food ingestion. A precise diagnostic test is essential to prevent T. gondii infection in both humans and animals. This study aimed to develop and evaluate the pETite-dense granule antigen 7(GRA7)-based indirect enzyme-linked immunosorbent assay (ELISA) to detect T. gondii infection in cats.Materials and methodsT. gondii-GRA7 was cloned and expressed in the Expresso®small ubiquitin-related modifier (SUMO) T7 Cloning and Expression System. The recombinant pETite-GRA7 was purified using HisTrap affinity chromatography and confirmed using Western blot analysis. The recombinant protein was used to develop and evaluate the indirect ELISA for T. gondii infection detection. In total, 200 cat sera were tested using pETite-GRA7-based indirect ELISA and indirect fluorescent antibody test (IFAT). The statistical analysis based on Kappa value, sensitivity, specificity, positive predictive value, negative predictive value, χ 2 test, and receiver operating characteristic (ROC) curve was used to evaluate the performance of the test.ResultsA 606 bp GRA7 polymerase chain reaction (PCR) product was obtained from T. gondii RH strain genomic DNA. The gene was cloned into the pETite™ vector and transformed to HI-Control Escherichia coli BL21 (DE3) for protein expression. Approximately 35 kDa of recombinant pETite-GRA7 was observed and Western blot analysis showed positive bands against anti-6-His antibody and positive-T. gondii cat serum. A sample of 0.5 μg/mL of pETite-GRA7 was subjected to indirect ELISA to detect T. gondii infection in the cat sera. The results showed sensitivity and specificity of pETite-GRA7-based indirect ELISA at 72% and 96%, respectively. An acceptable diagnostic performance was characterized by high concordant results (94%) and substantial agreement (Kappa value=0.65) with IFAT. The seroprevalence levels of ELISA and IFAT were 10% and 9%, respectively, and were not significantly (p>0.05) different. The expected performance of ELISA at different cutoff points using the ROC curve analysis revealed 89% sensitivity and 92% specificity at the cutoff value of 0.146, with a high overall assay accuracy (area under the curve=0.94).ConclusionIn this study, the pETite™ vector, N-terminal 6xHis SUMO fusion tag, was used to improve the solubility and expression level of GRA7. The recombinant pETite-GRA7 showed enhanced protein solubility and purification without special condition requirements. This pETite-GRA7-based indirect ELISA showed high concordant results and substantial agreement with IFAT. ELISA revealed an acceptable sensitivity and specificity. These initial data obtained from cats' sera demonstrated that pETite-GRA7-based indirect ELISA could be a useful method for local serological diagnosis of T. gondii infection in cats in Thailand.

Project description:BackgroundFree-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii.MethodsHere, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice.ResultsIn experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n = 61/65; or TgSAG1-ELISASH, n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7-99.9%) and specificity (100%; 95% CI: 85.0-100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 95% CI: 77.1-98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0-100%).ConclusionsThe TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

Project description:BackgroundTimely diagnosis of Toxoplasma gondii infection is necessary to prevent and control toxoplasmosis transmission. The gold immunochromatographic assay (GICA) is a means of rapidly detecting pathogen in samples. GICA-based diagnostic methods have been developed to accurately detect pathogens with high sensitivity and specificity, and their application in T. gondii diagnosis is expected to yield good results.MethodsColloidal gold test strips were produced using T. gondii C-terminal truncated apical membrane antigen 1 (AMA1C). Colloidal gold-AMA1C and colloidal gold-murine protein conjugate were synthesized under optimal conditions. A nitrocellulose membrane was treated with AMA1C and goat anti-mouse antibody as the test line and control line, respectively. In total, 90 cat serum samples were tested using AMA1C-GICA and a commercial enzyme linked immunosorbent assay (ELISA) kit. The GICA results were digitally displayed using a portable colloidal gold immunochromatographic test strip analyzer (HMREADER). The sensitivity, specificity, and stability of AMA1C-GICA were assessed, and this was then used to examine clinical samples, including 203 human sera, 266 cat sera, and 81 dog sera.ResultsAMA1C-GICA had a detection threshold of 1:32 for T. gondii-positive serum. The GICA strips specifically detected T. gondii antibodies and exhibited no reactivity with Plasmodium vivax, Paragonimus kellicotti, Schistosoma japonicum, Clonorchis sinensis, and Schistosoma mansoni. Consequently, 15 (16.7%) positive samples were detected using the AMA1C-GICA and commercial ELISA kits for each of the assays. The receiver-operating characteristic curve showed that GICA had a relative sensitivity of 85.3% and specificity of 92%, with an area under the curve of 98%. After analyzing clinical samples using HMREADER, 1.2%-23.4% of these samples were found to be positive for T. gondii.ConclusionsThis study presents a novel assay that enables timely and efficient detection of serum antibodies against T. gondii, thereby allowing for its early clinical diagnosis. Furthermore, the integration of digital detection using HMREADER can enhance the implementation of GICA.

Project description:Toxoplasma gondii evades host immunity to establish a chronic infection. Here, we assessed the role of parasitophorous vacuole (PV) membrane (PVM)- and intravacuolar network (IVN) membrane-localized dense granule (GRA) proteins in the development of acute and chronic Toxoplasma infection. Deletion of PVM-associated GRA3, GRA7, GRA8, and GRA14 or IVN membrane-associated GRA2, GRA9, and GRA12 in the low-virulence type II Prugniaud (Pru) strain induced severe defects in the development of chronic-stage cysts in vivo without affecting the parasite growth rate or the ability to differentiate into cysts in vitro Acute virulence of the Pru?gra2, Pru?gra3, and Pru?gra4 mutants was reduced but not abolished. In contrast, the Pru?gra12 mutant was avirulent in mice and Pru?gra12 parasites failed to establish a chronic infection. High-virulence type I strain RH?gra12 parasites also exhibited a major defect in acute virulence. In gamma interferon (IFN-?)-activated macrophages, type I RH?gra12 and type II Pru?gra12 parasites resisted the coating of the PVM with host immunity-related GTPases as effectively as the parental type I RH?ku80 and type II Pru?ku80 strains, respectively. Despite this resistance, ?gra12 PVs ultimately succumbed to IFN-?-activated host cell innate immunity. Our findings uncover a key role for GRA12 in mediating resistance to host IFN-? and reveal that many other IVN membrane-associated GRA proteins, as well as PVM-localized GRA proteins, play important roles in establishing chronic infection.IMPORTANCE Toxoplasma gondii cysts reactivate during immune deficiency and cause fatal encephalitis. Parasite molecules that coordinate the development of acute and chronic infection are poorly characterized. Here, we show that many intravacuolar network membrane and parasitophorous vacuole membrane-associated dense granule (GRA) proteins orchestrate the development of chronic cysts in vivo A subset of these GRA proteins also modulate acute virulence, and one protein that associates with the intravacuolar network membranes, namely GRA12, was identified as a major virulence factor required for parasite resistance to host gamma interferon (IFN-?). Our results revealed that many parasitophorous vacuole membrane and intravacuolar network membrane-associated GRA proteins are essential for successful chronic infection.

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Development of an immunochromatographic assay based on dense granule protein 7 for serological detection of Toxoplasma gondii infection.

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