Identification and real-time expression analysis of selected Toxoplasma gondii in-vivoinduced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients

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.

Seroprevalence of toxoplasma-specific antibodies in patients suspected to have active toxoplasmosis: A cross-sectional survey

“…Toxoplasma gondii is one of the most common intracellular protozoan parasites, with up to 30% of the world's human population affected by this parasite and the third main cause of food-related deaths in the United States of America[ 1 2 3 ]. In Iran, the rate of toxoplasmosis, in 2008, was 40.7% for Isfahan, 44.2% for Lorestan, and 34.2% for Bandar-e-Abbas.…”

“…[ 5 6 7 ] Humans are infected mainly by ingestion of T. gondii oocysts from contaminated soil, undercooked meat containing the parasite cysts, direct contact with cat feces or by vertical transmission from mother to fetus, via the placenta. [ 1 2 8 ] Although human toxoplasmosis is generally asymptomatic in a wide range of cases, it is of clinical importance when early infection occurs or when there is a reactivation of infection in immunosuppressed patients. [ 9 10 11 ] Toxoplasmosis commonly causes mild symptoms in immunocompetent individuals; whereas, in immunocompromised patients it is often fatal.…”

Immunoproteomic and mass spectrometric analysis of Eimeria acervulina antigens recognized by antisera from chickens infected with E. acervulina, E. tenella or E. necatrix

“…Toxoplasma gondii, an apicomplexan protozoan parasite, PP2C was identified as one of the highly upregulated genes in vivo by screening the T. gondii cDNA phage expression library using specific IgM and IgG [39]. The presence of upregulated immunogenic PP2C suggests that PP2C is a potential vaccine candidate for toxoplasmosis [40]. As a common antigen of Eimeria, GAPDH could induce strong humoral and cellular immune responses, and had effective protection against E. tenella, E. acervulina and E. maxima as a potential vaccine candidate [41].…”

Identification, production and assessment of twoToxoplasma gondiirecombinant proteins for use in aToxoplasmaIgG avidity assay

“…22 This library was previously screened with in vivo induced antigen technology (IVIAT) using pooled serum from individuals with suspected acute Toxoplasma infection and probed with anti-human IgG (HRP-conjugated). 23 Out of the many positive clones obtained, a total of 30 cDNA clones were randomly selected for use in the present study.…”

“…23,24 Briefly, Escherichia coli host cells XL1-Blue MRF' (Stratagene) were grown overnight in Luria-Bertani (LB) broth. The bacteria suspension was centrifuged and the cell pellet was resuspended in 10 mM MgSO 4 until the OD 600 reached 0.5.…”