Abstract
BackgroundToxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.MethodsThe pooled sera was first pre-absorbed against three different preparations of antigens from in-vitro-grown cells of each T. gondii and E. coli XL1-Blue MRF’, subsequently it was used to screen T. gondii cDNA phage expression library. Positive clones from each group were subjected to quantitative real-time PCR expression analysis on mRNA of in-vivo and in-vitro grown parasites.ResultsA total of 29 reactive clones from each IgM and IgG immunoscreenings were found to have high homology to T. gondii genes. Quantitative real-time PCR expression analysis showed that 20 IgM-detected genes and 11 IgG-detected genes were up-regulated in-vivo relative to their expression levels in-vitro. These included genes encoding micronemes, sterol-regulatory element binding protein site, SRS34A, MIC2-associated protein M2AP, nucleoredoxin, protein phosphatase 2C and several hypothetical proteins. A hypothetical protein (GenBank accession no. 7899266) detected by IgG had the highest in-vivo over in-vitro fold change of 499.86; while another up-regulated hypothetical protein (GenBank accession no. 7898829) recognized by IgM showed high sensitivity (90%) and moderate specificity (70%) in detecting T. gondii antibodies when tested with 20 individual serum samples.ConclusionThe highly up-regulated genes and the corresponding proteins, in particular the hypothetical proteins, may be useful in further studies on understanding the disease pathogenesis and as potential vaccine candidates.
Highlights
Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans
After three to four days postinfection, the peritoneal cavity fluid was aseptically harvested with 5 ml of RPMI-1640 medium containing penicillin streptomycin (RPMI-PS), pH 7.2 (Gibco®, Life Technologies, USA)
In our recent published report, T.gondii genes preferentially expressed in-vivo were identified via immunoscreening of T.gondii expression library with sera from chronicallyinfected individuals followed by real time polymerase chain reaction (PCR) to confirm the up-regulation of the identified genes
Summary
Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. Toxoplasmosis is caused by the zoonotic and ubiquitous Toxoplasma gondii, with up to 30% of the world human population affected by this parasite. Infection usually occurs by ingestion of T. gondii oocysts from infected cats or consumption of undercooked meat containing the parasite cysts. The foetus of an infected mother can acquire this infection by vertical transmission through the placenta during early pregnancy. Diagnosis of toxoplasmosis is usually performed by detection of IgG and IgM against T. gondii. IgG avidity test is an important additional test that is routinely performed; with low IgG avidity suggesting acute infection while high IgG avidity confirms chronic infection [9]
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