Toxoplasma gondii is an apicomplexan parasite with a broad host range of most warm-blooded mammals including humans, of which one-thirds of the human population has been infected worldwide which can cause congenital defects, abortion, and neonatal complications. Here, we developed a rapid diagnostic test (RDT) for T. gondii infection. Antigenic N-terminal half of the major surface antigen (SAG1) was linked with intrinsically unstructured domain (IUD) of dense granule protein 2 (GRA2). The recombinant GST-GRA2-SAG1A protein was successfully expressed and purified as 51 kDa of molecular weight. Furthermore, antigenicity and solubility of the rGST-GRA2-SAG1A protein were significantly increased. The overall specificity and sensitivity of GST-GRA2-SAG1A loaded RDT (TgRDT) were estimated as 100% and 97.1% by comparing with ELISA result which uses T. gondii whole cell lysates as the antigen. The TgRDT tested with Uganda people sera for field trial and showed 31.9% of seroprevalence against T. gondii antibody. The TgRDT is proved to be a kit for rapid and easy to use with high accuracy, which would be a suitable serodiagnostic tool for toxoplasmosis.

Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface. Molecular &

Rapid serodiagnostic methods for Toxoplasma gondii infection in cats are urgently needed for effective control of transmission routes toward human infections. In this work, 4 recombinant T. gondii antigens (SAG1, SAG2, GRA3, and GRA6) were produced and tested for the development of rapid diagnostic test (RDT). The proteins were expressed in Escherichia coli, affinity-purified, and applied onto the nitrocellulose membrane of the test strip. The recombinant SAG1 (rSAG1) showed the strongest antigenic activity and highest specificity among them. We also performed clinical evaluation of the rSAG1-loaded RDT in 182 cat sera (55 household and 127 stray cats). The kit showed 0.88 of kappa value comparing with a commercialized ELISA kit, which indicated a significant correlation between rSAG1-loaded RDT and the ELISA kit. The overall sensitivity and specificity of the RDT were 100% (23/23) and 99.4% (158/159), respectively. The rSAG1-loaded RDT is rapid, easy to use, and highly accurate. Thus, it would be a suitable diagnostic tool for rapid detection of antibodies in T. gondii-infected cats under field conditions.

Seroepidemiological changes of Toxoplasma gondii infection among the residents of the islands of Gangwha-gun, Incheon for 2 years were surveyed and evaluated by ELISA using a crude extract antigen. In 2010, sera of 919 adult residents in Gyodong-myeon and 313 adults in Samsan-myeon were collected and checked for IgG antibody titers, which showed 14.5% (133 sera) and 19.8% (62 sera) positive rates, respectively. In 2011, sera of 955 adults in Gyodong-myeon and 341 adults in Samsan-myeon were examined, which showed an increase of positive rates to 23.8% (227 sera) and 31.7% (108 sera), respectively. Totally, the seroprevalence of the first year was 15.8% and it increased rapidly to 25.8% in the second year. The positive rates of both sexes increased simultaneously with the significant ratio of males to females by 1.7-2.2 fold (P<0.05). In both myeons, 661 sera were collected every year and showed changes in optical density (OD) in 177 sera; newly found as positives in 73 persons (11.0%), negative conversion in 10 persons (1.5%), and maintained or increased in 94 persons (14.2%). This rapid increase in the prevalence of toxoplasmosis in Gangwha islands may be due to in part peculiar changes in the toxoplasmic environment of the islands and presumably the consumption of the pork bred domestically within the islands or imported from high endemic nations. It is necessary to find out symptomatic toxoplasmic patients and confirm the risk factors for further infection in the islands of Gangwha-gun.

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