| Toxoplasma gondii is an intracellular parasite that can infect domestic, wild, andcompanion animals, and it also commonly infects humans. The importance of thisparasite in food safety, human health and animal husbandry has been well recognized.Though T. gondii infection in humans with a normal immune competence isasymptomatic in most cases, the parasites do pose threats to individuals who areimmunocompromised, such as HIV carriers. It has been estimated that up to one thirdof the world’s population has been infected by T. gondii with an endemicity fromaround10%to70%and the prevalence is higher in warm and humid areas.Toxoplasma gondii displays significant genetic diversity in different geographicalregions, and the infection of Chinese people may be much more serious than weestimated. Thus it is critical to select accurate antigens to different diagnosis andepidemiological purposes.Currently, toxoplasmosis is diagnosed primarily by demonstratingparasite-specific IgM or IgG antibodies in serum samples. Most of the commerciallyavailable tests use T. gondii native antigens derived from the fast growing tachyzoiteswhich may result in variations in accuracy of detection. Recombinant antigens havebeen suggested as diagnostic reagents but their reliability may need extensiveexperimental validation. Further, T. gondii remains dormant as bradyzoites in immunecompetent individuals, which can convert to tachyzoites when the host immunedefense system is compromised, and tachyzoites and bradyzoites do display differentantigenic profiles. Thus it is critical to select accurate antigens for diagnostic andepidemiological purposes.In this study, we purified12recombinant antigens of T.gondii, and the overall sera-prevalence of anti-T. gondii IgG was performed by indirect ELISA assays. Weinvestigated more than800Chinese individuals using these12recombinant antigens,respectively. We also compared the sensitivity and consistency in detection of T.gondii specific antibodies in the same set of samples. Prevalence of T. gondii infectionin the Chinese population was systematically analyzed using the SPSS18.0softwarepackage.Firstly,12kinds of recombinant antigens were generated by affinitychromatography, which including tachyzoite-specific (SAG1, SAG5A and SAG5D),bradyzoite-specific (BAG1, BSR4and SRS9) and common antigens (MIC6, SRS4,GRA5, SUS1, SAG3, and SRS8) likely expressed in both tachyzoites and bradyzoites.In addition, the recombinant antigens from different strains were respectively namedas RH-rAg, ME49-rAg and RH/ME49-rAg. These antigens have been previouslyproved to be immunogenic and frequently recognized by human antibodies.SDS-PAGE and Western bloting results showed that molecular weights of theseproteins were consistent as expected. These recombinant proteins exhibited strongimmunogenic, since they have very stong recognition ability when incubating withGST-tag and His-tag mAbs, rat hyperimmune serum and human serum infectedT.gondii respectively.880serums samples from clinically healthy individuals were collected in2012.Indirect ELISA assays were performed to measure the levels of anti-T. gondii IgGantibodies in the sera according to the standard protocol. The purified recombinantprotein were treated as coating antigen for the serological test. The seroprevalence ofspecific IgG to the individual recombinant antigens were around10%, with oneexception, the positive rate (14.9%) of rBAG1was significantly higher than thatobserved with other recombinant antigens (p <0.05). Thus immuno-recognition ofBAG1is likely more prominent than other bradyzoite antigens during chronicinfection. BAG1was a30kDa cytosolic protein which is only expressed in thebradyzoites. Immunological studies suggested that BAG1was very immunogenic andcould induce early humoral and cell-mediated immune responses upon infection in humans. Thus it is not so surprising to observe a most prominent recognition ofBAG1by the sera compared to other tachyzoite-and bradyzoite-derived antigens.Analysis of the ELISA results in the assays with recombinant antigens of RH andME49strains showed, however, more overlapping between different strains. Amongthe248positive samples detected with the12recombinant antigens of either RH-,ME49-or common (RH/ME49) type,71samples were positive in reactions with allthree types of antigen, which accounted for28.6%of all positive samples, eventhough the detection rates with strain-specific rAgs were higher. The data furthersupported the finding that a combination of several immune-dominant antigens willgenerate a higher diagnostic efficiency. In contrast,6.9%of the positive sera sampleswere detected by both RH-rAg and ME49-rAg,11.3%were detected by both RH-rAgand common RH/ME49-rAg, and9.7%were detected by both ME49-rAg andRH/ME49-rAg. These results, again, suggested that the immune recognition of T.gondii antigens varied among different individuals.In this study, we compared the immunorecognition of three kinds of T. gondiistrain-and developmental status-specific antigens with the same set of sera. Resultsclearly showed that the prevalence of anti-T. gondii IgG obtained with recombinantantigens (rAgs) was significantly higher than that of the crude antigens previosly.More importantly, the number of sera that cross-reacted with the three kinds ofrecombinant antigens had shown a significantly higher consistency in detection of T.gondii-specific IgG in the serum samples. Thus, the general prevalence of anti-T.gondii IgG was likely much higher than previously reported. The data furthersupported the conclusion that there is more genetic diversity among the T. gondiiisolates in China, which argue for the necessity of the establishment of a method thatcan detect most, if not all, of the variant specific antibodies.Additionally, the ECM is composed of a complex assortment of glycoproteinsand proteoglycans. The ECM not only serves as scaffolding to provide a structuralframework for tissues but also regulates the behavior of the cells that contact it.Interactions between cells and ECM components affect cell adhesion, migration, proliferation and differentiation. The ECM may also serve as a binding site for thecolonization of microorganisms in the host. Microbial pathogens use adhesins to bindto ECM macromolecules such as fibronectin, collagen, vitronectin andthrombospondin. After binding to ECM proteins, microorganisms may use them as astronghold for propagating and spreading to other parts of the body. Thus, it is thoughtthat ECM components serve as docking sites for microbial pathogen invasion. ECMcomponents might participate actively in immune defense against microbial infection.It is reported that mindin is a highly conserved extracellular matrix (ECM)proein and is essential for the initiation of innate immune responses against bacterialpathogens. Mindin functions as a pattern-recognition molecule for microbialpathogens. Mindin-defcient mice exhibit an impaired ability to clear bacterialinfection, and mindin-defcient macrophages show defective responses to a broadspectrum of microbial stimuli. Moreover, mindin directly binds to bacteria and theircomponents and functions as an opsonin for the phagocytosis of bacteria. mindin alsoplays an essential role in the host innate immune response to infuenza virus infectionand suggest that mindin may be used as an immune-enhancing agent in infuenzainfection. In this study, recombinant antigens including rBAG1, rSRS4and rSRS9were used to immunized Balb/c mice, in combination with Freund’s adjuvant, mindinand PBS, respectively. The results exhibited that both Freund’s adjuvant and mindincould promote a strong humoral and cell-mediated immune responses. Moreover,these antigens were espected as vaccine candidate antigens of Toxoplasma gondii,Currently, the combined vaccine used for immuno-prevention has become verypromising. So, it is extremely important that seeking out T. gondii strain-anddevelopmental status-specific vaccine candidate antigens, which could produce thebetter protective effect. |
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